Droplet Assembly By 3D Printing

ABSTRACT

The invention relates to an apparatus for producing a droplet assembly, which apparatus comprises a droplet generator. A process for producing a droplet assembly, using an apparatus comprising a droplet generator is also described. The invention also relates to droplet assemblies comprising a plurality of droplets. Various uses of the droplet assemblies are also described.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/649,394, filed Jun. 3, 2015, which is the U.S. National Stage ofInternational Application No. PCT/GB2013/053229, file Dec. 6, 2013,which claims priority under 35 U.S.C. §§ 119 or 365(c), to Great BritainApplication No. 1222052.1, filed Dec. 7, 2012. The entire teachings ofthe above applications are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to an apparatus for producing a droplet assembly,which apparatus comprises a droplet generator. A process for producing adroplet assembly, using an apparatus comprising a droplet generator isalso described. The invention also relates to droplet assembliescomprising a plurality of droplets. Various uses of the dropletassemblies are also described.

BACKGROUND TO THE INVENTION

Aqueous droplets in a solution of lipids in oil adhere by forming lipidbilayers at their interfaces (Poulin, P. et al., Langmuir 14, 6341-6343(1998) and Holden, M. A., et al., J. Am. Chem. Soc. 129, 8650-8655(2007)). Networks of droplets functionalized with membrane proteins havebeen built and can act as light sensors (Holden, M. A., et al., J. Am.Chem. Soc. 129, 8650-8655 (2007)), batteries (Holden, M. A., et al., J.Am. Chem. Soc. 129, 8650-8655 (2007)) or simple electrical circuits(Maglia, G. et al., Nature Nanotech. 4, 437-440 (2009)). Dropletnetworks can be stabilized in bulk aqueous solution, and programmed torelease their contents upon a change in pH or temperature (Villar, G.,et al., Nature Nanotech. 6, 803-808 (2011)). However, defined networkshave been limited to simple arrangements of few droplets, typicallyassembled manually (Holden, M. A., et al., J. Am. Chem. Soc. 129,8650-8655 (2007) and Maglia, G. et al., Nature Nanotech. 4, 437-440(2009)), by microfluidic means (Bai, Y. et al., Lab. Chip 10, 1281-1285(2010), Zagnoni, M. et al., Lab. Chip 10, 3069-3073 (2010) and Stanley,C. E. et al., Chem. Commun. 46, 1620-1622 (2010)) or by manipulationwith external fields (Aghdaei, S., et al., Lab. Chip 8, 1617-1620(2008), Poulos, J. L., et al., Appl Phys Lett 95, 013706 (2009) andDixit, S. S., et al., Langmuir 26, 6193-6200 (2010)).

There is therefore an ongoing need to develop a method of producingcomplex droplet assemblies, such as a three-dimensional dropletassembly, and an apparatus for the production of a droplet assembly.

SUMMARY OF THE INVENTION

The present invention relates to an apparatus for producing a dropletassembly, which apparatus enables droplet assemblies to be produced byan automated process. The invention also relates to an automated processfor producing a droplet assembly.

The speed and precision with which a droplet assembly can be producedusing the apparatus of the invention and the process of the inventionallows complex assemblies of droplets to be created.

The inventors have demonstrated that millimetre-scale geometriescomprising tens of thousands of droplets can be produced. Even largerstructures with billions of droplets may also now be produced using thisinvention. Surprisingly, these structures are self-supporting andresistant to gentle perturbations. Further, the structures themselvesmay be complex and diverse, with structures not accessible by previouslyknown methods now being achievable.

The precise location and composition of each droplet in the dropletassembly can be controlled. Thus the droplet assemblies can be easilyfunctionalised, for instance, by the incorporation of membrane proteinsinto bilayers between specific contacting droplets. For example, theinventors have shown that a simple functional mimic of nervous tissuecan been produced by the inclusion of membrane proteins in specificdroplets within an assembly. The droplet assemblies may also befunctionalised by the inclusion of a variety of different materials,including small molecules, enzymes and living cells, within specificdroplets. Living cells may, for example, be allowed to grow within thedroplets of the droplet assembly, and to break down the bilayers betweendroplets some time after printing.

The droplet assemblies may also be used as sacrificial templates for thepatterning of solid materials. For example, inorganic materials may beincluded in specific droplets of the droplet assembly. Differentinorganic materials can be placed in different droplets within thedroplet assembly. The inorganic materials may then diffuse betweenspecific droplets, and react to form inorganic solids such as cadmiumsulphide.

Further, droplet assemblies can be built to comprise two or moredifferent compartments. The individual compartments may communicate witheach other and/or with the external environment by, for example, usingmembrane proteins. Sophisticated, compartmentalised systems maytherefore be produced.

Utilising the process of osmosis, the inventors have also been able tocreate self-folding networks that fold in a predictable way. The abilityof the droplet assembly to change shape allows new and moresophisticated structures to be developed. The assemblies could, forexample, be designed as a hydraulic mimic of muscle tissue. Applicationssuch as the use of a droplet assembly as an autonomously functioningentity, interacting with living organisms or electronics, are now arealistic prospect. An assembly may, for example, be used as a platformfor drug delivery or even as part of an artificial tissue. The use ofdroplet networks in tissue engineering is a particularly interestingprospect as it could reduce or even overcome many issues commonlyobserved with living cells, such as the replication and migration ofcells and the rejection of tissues by the body.

Accordingly, the invention provides an apparatus for producing a dropletassembly, which apparatus comprises: at least one droplet generator; acontainer which is moveable relative to the at least one dropletgenerator; and a control unit, which control unit is adapted to controlthe dispensing of droplets from the at least one droplet generator andthe movement of the container relative to the at least one dropletgenerator, wherein the apparatus is adapted to produce a dropletassembly which comprises a plurality of droplets, wherein each of saiddroplets comprises (i) a droplet medium and (ii) an outer layer ofamphipathic molecules around the surface of the droplet medium, whereinthe droplet medium is an aqueous medium or a hydrophobic medium, andwherein at least one of said droplets contacts another of said dropletsto form a layer of said amphipathic molecules as an interface betweenthe contacting droplets.

The invention also provides a process for producing a droplet assemblyusing an apparatus for producing the droplet assembly, which dropletassembly comprises: a plurality of droplets, wherein each of saiddroplets comprises: (i) a droplet medium, and (ii) an outer layer ofamphipathic molecules around the surface of the droplet medium, whereinthe droplet medium is an aqueous medium or a hydrophobic medium, andwherein at least one of said droplets contacts another of said dropletsto form a layer of said amphipathic molecules as an interface betweenthe contacting droplets; which apparatus comprises: at least one dropletgenerator; a container which is moveable relative to the at least onedroplet generator; and a control unit, which control unit is adapted tocontrol the dispensing of droplets from the at least one dropletgenerator and the movement of the container relative to the at least onedroplet generator; wherein said container of the apparatus contains abulk medium, wherein: when the droplet medium is an aqueous medium thebulk medium is a hydrophobic medium, and when the droplet medium is ahydrophobic medium the bulk medium is an aqueous medium; which processcomprises: (a) a plurality of dispensing steps, wherein each dispensingstep comprises dispensing a droplet of the droplet medium from a saiddroplet generator into the bulk medium, in the presence of amphipathicmolecules, and thereby forming in the bulk medium a droplet whichcomprises (i) said droplet medium and (ii) an outer layer of amphipathicmolecules around the surface of the droplet medium; and (b) moving thecontainer relative to the at least one droplet generator, to control therelative positioning of the droplets in the bulk medium.

In another aspect, the invention provides a droplet assembly which isobtainable by a process as defined hereinabove.

In a further aspect, the invention provides a droplet assembly whichcomprises a plurality of droplets, wherein each of said dropletscomprises (i) an aqueous medium, and (ii) an outer layer of amphipathicmolecules around the surface of the aqueous medium, and wherein each ofsaid droplets contacts another of said droplets to form a bilayer ofsaid amphipathic molecules as an interface between the contactingdroplets, wherein the plurality of droplets comprises a first region ofsaid droplets and a second region of said droplets, wherein each dropletin the first region contacts at least one other droplet in the firstregion to form a bilayer of said amphipathic molecules as an interfacebetween the contacting droplets, and each droplet in the second regioncontacts at least one other droplet in the second region to form abilayer of said amphipathic molecules as an interface between thecontacting droplets, wherein the aqueous medium of the droplets in thefirst region has a first osmolarity and the aqueous medium of thedroplets in the second region has a second osmolarity, wherein the firstosmolarity is different from the second osmolarity.

Also provided by the invention is a droplet assembly which comprises aplurality of droplets, wherein each of said droplets comprises (i) anaqueous medium, and (ii) an outer layer of amphipathic molecules aroundthe surface of the aqueous medium, and wherein each of said dropletscontacts another of said droplets to form a bilayer of said amphipathicmolecules as an interface between the contacting droplets, wherein theplurality of droplets defines a shell around a volume within the dropletassembly that does not comprise said droplets.

In further aspects, the invention provides various uses of the dropletassemblies of the invention as defined herein.

Thus, the invention provides the use of a droplet assembly of theinvention as defined herein in synthetic biology.

The invention also provides the use of a droplet assembly of theinvention as defined herein as a drug-delivery vehicle.

The invention also provides the use of a droplet assembly of theinvention as defined herein in tissue engineering. A droplet of thedroplet assembly may, for instance, comprise living cells.

The invention also provides the use of a droplet assembly of theinvention as defined herein in material science and engineering.

Further provided by the invention is the use of a droplet assembly asdefined herein for the droplet assembly of the invention as a templatefor the patterning of a solid material.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic of a droplet generator. The droplet generatorchamber is filled with a volume V of aqueous solution. The nozzle oflength L has an internal diameter D at its base, and a tip diameter d.The level of the aqueous solution in the chamber is a distance h abovethe level of the oil solution in the well.

FIGS. 2A-2B show printed droplet networks. FIG. 2A shows a schematic ofa setup of the apparatus. Two droplet generators eject droplets ofdifferent aqueous solutions into a well filled with a solution of lipidsin oil. The well is mounted on a motorized micromanipulator. The dropletgenerators and the manipulator are controlled by a personal computer(PC). FIG. 2B provides a schematic of a droplet network being printed.Aqueous droplets ejected into the oil acquire a lipid monolayer, andform bilayers with droplets in the growing network.

FIG. 2C shows images that define the desired horizontal cross-sectionsof a three-dimensional droplet network. The design comprises 20 layersof 50×35 droplets each; only alternate cross-sections are shown. In FIG.2D, a network printed according to the design in FIG. 2C is shown. Thescale bar represents 5 mm.

FIG. 2E is a schematic of another three-dimensional design, whichconsists of 28 layers of 24×24 droplets each. FIG. 2F shows threeorthogonal views of a single network printed according to the design inFIG. 2E. The scale bar represents 1 mm.

FIG. 3 provides a diagram of a droplet generator. The device consists ofa piezoelectric transducer affixed onto a micromachined poly(methylmethacrylate) chamber. A rubber septum is fitted opposite thetransducer, and into the septum is inserted a pulled and bent glasscapillary. The two lower holes were used to fix one device in place, andto mount the other onto a manual micromanipulator. All dimensions are inmm.

FIG. 4 provides a schematic of driving electronics for dropletgenerators. The electronics allow a computer to apply a voltage in therange −30 V to +30 V, with 12-bit resolution, to one of twopiezoelectric transducers. Abbreviations: PC, personal computer; USB,universal serial bus; SPI, serial peripheral interface; DAC,digital-to-analog converter; op amp, operational amplifier.

FIG. 5 shows a graphical user interface. The left and centre panelsallow, for each of the two generators, variation of the duration (in μs)and amplitude (as a proportion of 60 V) of the voltage pulse, thetriggering of droplet ejection, variation of the number of droplets tobe ejected per trigger, and variation of the delay (in ms) betweenmultiple droplets made per trigger. The right panel allows interactivecontrol of the manipulator, and variation of the manipulator step sizeand speed.

FIGS. 6A-6B show a printing pattern. FIG. 6A provides an example of amap of 49 pixels, including A (light grey), B (dark grey, in the centreof the diagram) and empty (black) pixels. FIG. 6B shows the path takenby the printing nozzles across the map in each of the two passes, in thesimplest variation of the printing algorithm.

FIGS. 7A-7C demonstrate printing distortions. All the networks in thisfigure were printed with the simple printing pattern of FIG. 6B. FIG. 7Ashows a horizontal view of a network designed to be cuboidal, butresulting in a convex top face. FIG. 7B shows a view from above of anetwork designed to be cuboidal, but resulting with inwardly slopedwalls. FIG. 7C shows a view from above of a network designed to becuboidal and faithfully printed by the simple printing pattern.

FIG. 8 provides an outline of a printing algorithm. The instructionslabelled Initialization are executed first, followed by those labelledDirection. The conditions marked by squares must be met before thealgorithm continues to the next instruction. At the evaluation of theconditions marked by diamonds, the algorithm continues to theinstruction marked by the horizontal arrow if the condition is true, orto that marked by the vertical arrow if the condition is false. Thedelays after making each droplet or row are explained in the Examples,under Supplementary Discussion.

FIG. 9 shows an electrical representation of a general droplet network.V_(i) and V_(j) are the electrical potentials at the droplets labelled iand j, respectively. c_(ij) and r_(ij) are, respectively, thecapacitance and resistance between droplets i and j. I_(ij) is the ioniccurrent owing from j into i, and consists of a capacitive componentI_(ij) ^(c) and resistive component I_(ij) ^(r). The droplets areillustrated on a square grid only for clarity; the derivation in theExamples applies to a network in any arrangement.

FIG. 10 demonstrates droplet ejection as a function of pulse width andvoltage. Photographs were taken immediately after droplet ejection, forvarious widths and amplitudes of the voltage pulse applied to a dropletgenerator. To determine the reproducibility of the ejected droplets,each combination of pulse width and amplitude was applied five timeswith an interval of a few seconds between pulses. After everycombination was tested, the entire procedure was repeated. Dropletproduction was consistent for each combination of pulse width andamplitude (n=10) for a given nozzle, but varied between nozzles. Thehighlighted photographs indicate conditions for this nozzle thatproduced single droplets of a suitable size for printing dropletnetworks. The scale bar represents 200 μm.

FIGS. 11A-11B show droplet networks printed in bulk aqueous solution.FIG. 11A provides a schematic of printing in aqueous solution. Thenozzles eject aqueous droplets into a drop of oil that is suspended inbulk aqueous solution by a wire frame. FIG. 11B shows a micrograph of anetwork printed in aqueous solution, viewed from above. A core of lightgrey droplets is surrounded by a shell of darker grey droplets, whichcontain the fluorescent dye pyranine. The scale bar represents 400 μm.

FIG. 11C shows horizontal sections of the network in FIG. 11B obtainedby confocal microscopy, showing the fluorescent shell of droplets aroundthe non-fluorescent core. The sections span approximately the bottom 150μm of the network. The scale bar represents 400 μm.

FIG. 11D shows micrographs of three other networks printed in bulkaqueous solution. The scale bars represent 400 μm.

FIG. 12A demonstrates an electrically conductive pathway. FIG. 12A showsa schematic of part of a network printed with a pathway that allows theflow of ionic current. Only the light grey droplets and the large dropcontain α-hemolysin (αHL) pores. The large drop is impaled with anAg/AgCl electrode. The magnified section illustrates the αHL pores inthe bilayers around the αHL-containing droplets.

FIG. 12B provides a photograph of a printed network withelectrode-impaled drops placed on either end of the conductive pathway.The light grey droplets contain αHL, while the other droplets contain noprotein. The scale bar represents 500 μm. FIG. 12C shows a stepwiseincrease in the ionic current, as measured in the configuration in FIG.12B, at 50 mV in 1 M KCl at pH 8.0.

FIG. 12D provides a photograph of the network in FIG. 12B, afterseparating one of the large drops and rejoining it onto the network at aposition away from the pathway. The scale bar represents 500 μm. FIG.12E shows selected portions of a single recording as measured in theconfiguration in d at 50 mV, showing transient increases in ioniccurrent.

FIGS. 13A-13B illustrate electrical measurements of droplet networkswith and without αHL. FIG. 13A shows a photograph of a network in whichnone of the droplets contained αHL. The network droplets and the largeelectrode-impaled drops contained 25 mM Tris(tris(hydroxymethyl)aminomethane) HCl, 1 M KCl, 100 μM EDTA, pH 8.0. Thenetwork droplets additionally contained 1 mM xylene cyanol FF, and theelectrode-impaled drops additionally contained αHL and 10 mM pyranine.The scale bar represents 500 μm. FIG. 13B provides a typical portion ofthe current measured at 50 mV in the configuration shown in FIG. 13A. Nosteps or transient spikes of current were measured from this network inany recording.

FIG. 13C provides a photograph of a network in which all of the dropletscontained αHL. The network droplets and the electrode-impaled drops wereof the same solution as the electrode-impaled drops in FIG. 13A. Thescale bar represents 500 μm. FIG. 13D shows a portion of the currentmeasured at 150 mV in the configuration shown in FIG. 13C, immediatelyafter the large drops were placed onto the network. Similar currentsteps were measured after twice removing and replacing the large dropsonto the network.

FIGS. 14A-14B demonstrate electrical simulations of a conductive dropletpathway. FIG. 14A provides a schematic of the system simulated as amodel of the electrical recording conditions in FIG. 12B. The networkconsists of 4 rows, 20 columns and 4 layers of droplets in aface-centred cubic arrangement, and represents only the conductivepathway part of the network in FIG. 12. Two large drops are positionedat either end of the network, and form bilayers with the networkdroplets at the end of each row. FIG. 14B shows a simulated currentbetween the electrodes in the system depicted in FIG. 14A. Thesimulation used the following parameter values, as an approximation ofthe conditions of the experiments described in the Examples: an appliedpotential of 50 mV, bilayer diameters of 45 μm, a bilayer specificcapacitance of 650 nF cm⁻² (Holden, M. A., et al., J. Am. Chem. Soc.129, 8650-8655 (2007)), a bilayer conductance of 1 pS, and a poreconductance of 1 nS (Holden, M. A., et al., J. Am. Chem. Soc. 129,8650-8655 (2007)). Five αHL pores are assumed to be initially present ineach bilayer within the network. Each of the bilayers between thenetwork and drop a is assumed to initially contain one pore, while thosebetween the network and drop b are assumed to contain no pores. Theinsertion of a single αHL pore into a bilayer between the network anddrop a is simulated to occur at 1 s, 3 s, 5 s and 7 s (dark greyarrows), and into a bilayer between the network and drop b at 2 s, 4 s,6 s and 8 s (light grey arrows). Each pore inserts into a differentbilayer. Note that to decrease the computation time, the time intervalof the simulation was made shorter than that in FIG. 12C.

FIG. 14C shows a schematic of the system simulated as a model of theelectrical recording conditions in FIG. 12D. The network is identical tothat in FIG. 14A, except for the addition of two columns of dropletsthat do not contain αHL. FIG. 14D shows a simulated current between theelectrodes in FIG. 14C. The simulation was performed with the sameconditions as described in FIG. 14B, except that no pores were initiallypresent in any of the bilayers formed by the rightmost column ofdroplets in the network. The arrows signify pore insertions as describedin FIG. 14B.

FIGS. 15A-15C illustrate self-folding networks. FIG. 15A provides aschematic of two droplets of different osmolarities joined by a lipidbilayer. The transfer of water through the bilayer causes the dropletsto swell or shrink. FIG. 15B shows a schematic of a droplet network thatcomprises two strips of droplets of different osmolarities. The transferof water between the droplets induces an overall deformation of thenetwork. FIG. 15C provides photographs of a rectangular network foldinginto a circle over ˜3 h. The light grey and dark grey droplets initiallycontain 250 mM KCl and 16 mM KCl, respectively. The scale bar represents250 μm.

FIG. 15D shows photographs of a flower-shaped network foldingspontaneously into a sphere. The light grey and darker grey dropletsinitially contain 80 mM KCl and 8 mM KCl, respectively. The photographscover a period of 8 h. The scale bar represents 200 μm. FIG. 15E showsthe network in FIG. 15D in its final configuration, photographed fromabove. The scale bar represents 200 μm. FIG. 15F shows frames from afolding simulation of a network with a similar initial geometry to thenetwork shown in FIG. 15D. Dark grey (top layer) and light grey (bottomlayer) represent the lowest and highest initial osmolarities,respectively, and white (final frame) indicates the average of the two.

FIGS. 16A-16B illustrate the measurement of water permeability ofdroplet interface bilayers. FIG. 16A provides photographs of a singlepair of droplets with different salt concentrations, taken every 20 minafter the droplets were joined by a bilayer. The dark grey dropletcontained 25 mM Tris-HCl, 1 M KCl, 100 μM EDTA, pH 8.0. The lighter greydroplet contained the same solution, diluted to obtain a saltconcentration of 250 mM KCl. The dark grey droplet additionallycontained 1 mM xylene cyanol FF, and the lighter grey (smaller) dropletadditionally contained 2.5 mM orange G. The scale bar represents 1 mm.FIG. 16B demonstrates the volumes of three pairs of droplets asexemplified in FIG. 16A. The droplet and bilayer diameters were measuredfrom photographs, and the droplet volumes calculated with the assumptionof spherical cap geometries. The dark grey and light grey circles showthe measured volumes of the dark grey and light grey droplets,respectively. These data were used to calculate the permeabilitycoefficient P as described in the Examples, under Supplementary Methods.The dark grey and light grey lines show the droplet volumes calculatedusing Eq. (9) in the Examples, under Supplementary Methods, with theaverage measured value of P=27 μm s⁻¹. The calculated curves demonstratethat the data are well explained by the model with the average value ofP.

FIGS. 17A-17C demonstrate fracture of folding networks. FIG. 17A shows anetwork composed of two strips of droplets similar to that in FIG. 15C,except that the strip of droplets of lower osmolarity in this networkwas thinner horizontally. In FIG. 17B the same network as in FIG. 17A isdepicted, after the completion of folding. The point of fracture in theregion of dark grey droplets is evident. FIG. 17C shows an originallycross-shaped network similar to the petal-shaped network in FIGS. 15Dand 15E. The layer of lower osmolarity fractured near the base of theupper arm, as evidenced by the absence of dark grey droplets in thatregion. Note that the fractured arm folded to a lesser extent than theothers.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an apparatus for producing a dropletassembly, which apparatus comprises: at least one droplet generator; acontainer which is moveable relative to the at least one dropletgenerator; and a control unit, which control unit is adapted to controlthe dispensing of droplets from the at least one droplet generator andthe movement of the container relative to the at least one dropletgenerator, wherein the apparatus is adapted to produce a dropletassembly which comprises a plurality of droplets, wherein each of saiddroplets comprises (i) a droplet medium, and (ii) an outer layer ofamphipathic molecules around the surface of the droplet medium, whereinthe droplet medium is an aqueous medium or a hydrophobic medium, andwherein at least one of said droplets contacts another of said dropletsto form a layer of said amphipathic molecules as an interface betweenthe contacting droplets.

Typically, the apparatus is adapted to produce said droplet assemblywherein each of said droplets contacts another of said droplets to forma layer of said amphipathic molecules as an interface between thecontacting droplets.

The layer of said amphipathic molecules which is an interface betweencontacting droplets may, for instance, be a bilayer of said amphipathicmolecules or a layer of a block copolymer. The layer of a blockcopolymer may, for example, be a layer of a triblock copolymer.

When the droplet medium is an aqueous medium the layer of saidamphipathic molecules which is an interface between contacting dropletsmay, for instance, be a bilayer of said amphipathic molecules or a layerof a block copolymer.

When the droplet medium is a hydrophobic medium the layer of saidamphipathic molecules which is an interface between contacting dropletsmay, for instance, be a bilayer of said amphipathic molecules or a layerof a block copolymer. In the bilayer of amphipathic molecules formed,the polar heads of the amphipathic molecules are in contact with eachother. (See, for example, Aronson et al., Nature, Vol. 286, July 1980and in Poulin et al., J. Phys. Chem. B, Vol. 103, no. 25, June 1999.)

Typically, the layer of said amphipathic molecules which is an interfacebetween contacting droplets is a bilayer of said amphipathic molecules.

The droplet medium is typically an aqueous medium. Another embodiment isenvisaged however in which the droplet medium is a hydrophobic medium.

Usually, the droplet medium is an aqueous medium. The aqueous medium maybe any suitable aqueous medium. For instance, the aqueous medium may bepure water, or an aqueous buffer solution, or an aqueous solution of oneor more salts. Alternatively, the aqueous medium may comprise ahydrogel. When the aqueous medium comprises a hydrogel, the aqueousmedium may, for instance, comprise agarose and water. The concentrationof the agarose in water is typically less than or equal to 10% w/vagarose. For instance, the concentration of the agarose in said watermay be from 0.25 to 5% w/v agarose. Hydrogels other than agarose mayalso be used. For instance the aqueous medium may comprisemethylcellulose, polyethylene glycol diacrylate, polyacrylamide,matrigel, hyaluronan, polyethylene oxide, polyAMPS(poly(2-acrylamido-2-methyl-1-propanesulfonic acid)),polyvinylpyrrolidone, polyvinyl alcohol, sodium polyacrylate, acrylatepolymers or poly(N-isopropylacrylamide). Alternatively, the aqueousmedium body may comprise a silicone hydrogel or LB (Luria broth) agar.

One important property of the aqueous medium is pH and this can bevaried over a wide range. In some embodiments, for instance, the pH ofthe aqueous medium within the aqueous droplet or droplets may be in therange of from 5 to 9 (or for instance in the range of from 6 to 8)although higher and lower pH values are also possible. The aqueousmedium may therefore be an aqueous buffer solution. Any suitable buffercan be employed, depending on the desired pH. The buffer solution mayfor instance comprise Tris-HCl and/or KCl. In some embodiments the pH ofthe aqueous buffer solution is from 5 to 9, or for instance from 6 to 8.The nature and concentration of the solutes can be varied to vary theproperties of the solution.

The aqueous medium of each droplet in the droplet assembly may be thesame or different.

The amphipathic molecules of a droplet need not be all of the same type.Rather, the amphipathic molecules may in some embodiments be a mixtureof two or more different kinds of amphipathic molecule. Anotherimportant example is that the amphipathic molecules in the respectiveouter layers of different droplets in a droplet assembly may be ofdifferent types so that the bilayer(s) formed between the differentdroplets may be asymmetric. Typically, the apparatus is adapted toproduce a droplet assembly which is disposed in a bulk medium whereinwhen the droplet medium is an aqueous medium the bulk medium is ahydrophobic medium, and when the droplet medium is a hydrophobic mediumthe bulk medium is an aqueous medium. When the bulk medium is an aqueousmedium, the aqueous medium may be as further defined hereinbefore.Similarly, when the droplet medium is a hydrophobic medium, thehydrophobic medium may be as further defined hereinbelow.

Usually, the droplet medium is an aqueous medium and the bulk medium isa hydrophobic medium, and the invention will generally be describedhereinbelow in these terms. However, as the skilled person willappreciate, any of the embodiments of the invention described herein inthose terms may also be performed “in reverse”, using a hydrophobicmedium as the droplet medium instead of an aqueous medium, and using anaqueous medium as the bulk medium instead of a hydrophobic medium.

Typically, the droplet medium is an aqueous medium and the apparatus isadapted to produce a droplet assembly which is disposed in a hydrophobicmedium. The hydrophobic medium may, for instance, be a hydrophobicmedium as further defined hereinbelow.

A droplet of the aqueous medium is usually dispensed into thehydrophobic medium in the presence of amphipathic molecules. Theamphipathic molecules may, for instance, be disposed in the aqueousmedium or in the hydrophobic medium. Typically, the amphipathicmolecules are disposed in the hydrophobic medium.

When the aqueous medium is dispensed into the hydrophobic medium in thepresence of amphipathic molecules, an aqueous droplet forms, whichdroplet comprises (i) an aqueous medium and (ii) an outer layer ofamphipathic molecules around the surface of the aqueous medium.

The droplet assembly comprises at least two droplets in contact witheach other. The boundary that is shared between contacting droplets, atthe point of contact between the objects, is referred to herein as aninterface. An interface is formed when part of the outer layer of onedroplet contacts part of the outer layer of another droplet. Forinstance, when the droplet is brought into contact with the otherdroplet, a bilayer of amphipathic molecules may form at the interfacebetween the two objects. The bilayer comprises amphipathic moleculesfrom the outer layer of amphipathic molecules around the surface of theaqueous medium of each droplet at the interface. The bilayer forms as itis an energetically more favourable configuration for the amphipathicmolecules to adopt. The contacting droplets will acquire the geometrywith the lowest free surface energy.

Typically, the apparatus of the invention is adapted to produce saiddroplet assembly wherein each of said droplets contacts another of saiddroplets to form a bilayer of said amphipathic molecules as an interfacebetween the contacting droplets.

The droplet assembly may comprise one interface, or it may comprise twoor more interfaces. Typically, the droplet assembly comprises at least nof said droplets, and at least n−1 of said interfaces between contactingdroplets, wherein n is equal to or greater than 2. The integer n may beequal to or greater than 3. More typically, n is equal to or greaterthan 4.

In some embodiments, when the droplet assembly comprises at least n ofsaid droplets, the network may comprise n or more than n interfaces,wherein n is as herein defined, it being understood that any one dropletcan be in contact with (and therefore form an interface with) more thanone other droplet.

An advantage of the present invention is that the apparatus enablesmillimetre-scale, or larger, droplet assemblies to be produced. Theinteger n can in principle be very high, for instance of the order ofmillions. Such networks, which can in principle comprise millions ofdroplets, may, for instance, be useful for preparing prototissue (i.e.an multi-compartment analogue of protocells) or minimal tissue. In someembodiments, therefore, the integer n may be as high as several million,for instance up to about 100,000,000, or for instance up to about50,000,000. The integer n may, for instance, be up to about 10,000,000,or for instance up to about 5,000,000.

In other embodiments, n may be at least several hundred, for instance atleast about 500, or for instance at least about 1000. The integer n mayfor instance be an integer of from 500 to 5,000,000, or an integer offrom 5,000 to 500,000. n may be an integer of from 10,000 to 50,000.

The droplet assembly may, for instance, be a droplet assembly as definedhereinbelow.

Droplet assemblies produced using the apparatus of the invention may bemulti-compartment systems. The droplet assembly may, for instance,comprise a first compartment and a second compartment. The firstcompartment within the droplet assembly may communicate with the secondcompartment via membrane proteins. The first and/or second compartmentmay communicate with the external environment (i.e. environment externalto the droplet assembly) via membrane proteins. In principle, a dropletassembly may comprise a large number of compartments and architecturallydefined structures may thus be produced.

The control unit of the apparatus is usually adapted to coordinate (a)the movement of the container relative to the or each droplet generatorand (b) the dispensing of the droplets, to create said droplet assembly.

The control unit may, for instance, be adapted to control the movementof the container. Alternatively, the control unit may be adapted tocontrol the movement of the or each droplet generator. Typically, thecontrol unit is adapted to control the movement of the container.

Typically, the control unit comprises a computer or dedicated electronichardware. More typically, the control unit comprises at least onecomputer. It usually comprises at least one personal computer (PC). Thecontrol unit may, for instance, be a PC. In some embodiments, thecontrol unit comprises a PC and dedicated electronic hardware (as shownin FIG. 4).

Usually, the control unit is adapted to control the dispensing ofdroplets of the aqueous medium from the or each droplet generator. Anexample of the driving electronics for the or each droplet generator isprovided in FIG. 4.

The droplet generator may, for instance, be a microfluidic system. Itmay, for instance, (a) generate droplets on demand or (b) producedroplets in a continuous stream and select specific droplets to bedeposited. Usually, the droplet generator generates droplets on demand.The droplet generator may: (i) generate and expel a droplet in a singlestep; or (ii) generate and expel a droplet in separate steps. Usually,the droplet generator generates and expels a droplet in a single step.

Typically, the at least one droplet generator is a piezoelectric dropletgenerator. More typically, the at least one droplet generator is apiezoelectric droplet generator which comprises a piezoelectrictransducer for dispensing droplets.

The aqueous medium may, for instance, be dispensed from the or eachdroplet generator by the application of a voltage pulse to thepiezoelectric component. Usually, the control unit controls theapplication of the voltage pulses to the piezoelectric component.Typically, the voltage pulse has a peak-to-peak amplitude of from 5 V to100 V, for instance, of from 10 V to 80 V. The peak-to-peak amplitudemay, for instance, be of from 20 V to 60 V. Typically, each pulse has aduration of from 10 to 1,500 μs, for instance, of from 50 to 1,000 μs.More typically, each pulse has a duration of from 100 to 800 μs.Usually, the voltage pulse is a square voltage pulse.

As shown in FIG. 10, the diameter of the droplet may be tuned by varyingthe amplitude and duration of the voltage pulses. By varying theseparameters, the droplet diameter can be tuned to a suitable diameter.The diameter may, for instance, be tuned to be from about 10 to 200 μm.Thus the control unit may be adapted to control the application of afirst voltage pulse and a second voltage pulse where the first voltagepulse and the second voltage pulse are different. A single dropletgenerator can thus be used to produce droplets of different sizes.Alternatively, the first and second voltage pulses could be applied tothe piezoelectric component of two different droplet generators.

The peak-to-peak amplitude defines the absolute value of the differencebetween the peak (or highest voltage) and the trough (or lowest value).

The size of the droplets produce may be controlled by adapting thedroplet generator. As illustrated in FIGS. 1 and 3, the dropletgenerator may comprise an outlet. The outlet may be adapted to controlthe size of the droplets produced. The outlet is discussed furtherbelow.

Typically, the control unit is adapted to control the dispensing ofdroplets of the aqueous medium from the or each droplet generator, sothat droplets are dispensed at a rate of from 0.01 to 100 s⁻¹, forinstance, at a rate of from 0.01 to 50 s⁻¹. Usually, the control unit isadapted to control the dispensing of droplets of the aqueous medium fromthe or each droplet generator, so that droplets are dispensed at a rateof from 0.01 to 10 s⁻¹.

Usually, the apparatus comprises two or more of said droplet generators.Typically, each droplet generator is as herein defined.

The size of a droplet produced may be controlled by adapting the dropletgenerator. Having two or more of said droplet generators enablesdifferently-sized droplets to be produced.

The two or more droplet generators may dispense the same aqueous mediumor the two or more droplet generators may dispense different aqueousmedia. Therefore, having two or more of said droplet generators allowsdroplets comprising different aqueous media to be produced. Forinstance, a droplet assembly comprising a first droplet comprising afirst aqueous medium and a second droplet comprising a second aqueousmedium may be produced using the apparatus of the invention.

Having two or more of said droplet generators allows, for example, (i)differently sized droplets to be produced and/or (ii) dropletscomprising different aqueous media to be produced. Differently sizeddroplets may, for instance, be produced by applying different voltagesto the piezoelectric components of the different droplet generators orby adapting the droplet generator.

When the amphipathic molecules are disposed in the aqueous medium,droplets comprising different amphipathic molecules may, for instance,be produced. For instance, a first droplet generator may comprise afirst aqueous medium comprising a first amphipathic molecule and asecond droplet generator may comprise a second aqueous medium comprisinga second amphipathic molecule.

The apparatus of the invention usually further comprises amicromanipulator for moving the container relative to the or eachdroplet generator. Typically, the control unit is adapted to controlmovement of the container relative to the or each droplet generatorusing the micromanipulator.

More typically, the apparatus of the invention further comprises amicromanipulator for moving the container, and wherein the control unitis adapted to control movement of the container using themicromanipulator.

The micromanipulator is generally a motorized micromanipulator.

The container is typically disposed on the micromanipulator, so that themovement of the micromanipulator causes movement of the container.Typically, the control unit is adapted to control movement of themicromanipulator, which in turn causes movement of the container.

The control means is typically adapted to communicate with themicromanipulator for moving the container via an electrical or wirelesssignal. The control means is typically in electrical connection with themicromanipulator. Alternatively, the control means is capable ofcommunicating with the micromanipulator wirelessly.

In some embodiments, the apparatus further comprises a micromanipulatorfor moving the or each droplet generator and, when the apparatuscomprises more than one said droplet generator, for coordinating therelative displacement of the droplet generators.

Individual droplet generators may, for instance, be moved together orseparately.

The micromanipulator for moving the container may be the same asmicromanipulator for moving the or each droplet generator.Alternatively, the micromanipulator for moving the container may be thedifferent from micromanipulator for moving the or each dropletgenerator. Typically, the micromanipulator for moving the container isthe same as micromanipulator for moving the or each droplet generator.

The control means is typically adapted to communicate with themicromanipulator via an electrical or wireless signal. The control meansis typically in electrical connection with the micromanipulator.Alternatively, the control means is capable of communicating with themicromanipulator wirelessly.

Typically, the or each droplet generator comprises a chamber for holdinga droplet medium (typically an aqueous medium); an outlet; and acomponent for displacing a volume of said droplet medium through saidoutlet and thereby dispensing said volume as a droplet.

In some embodiments, the or each droplet generator further comprises aninlet.

Typically, the inlet allows entry of air into the chamber of the dropletgenerator upon the dispensing of a droplet from the outlet.

The inlet may, for instance, be for introducing an aqueous medium intothe chamber. The chamber is typically filled with from 200 to 600 μl ofthe aqueous medium, for instance from 400 to 500 μl. For instance, thechamber may be filled with about 400 μl of the aqueous medium. Usually,the chamber is filled with the aqueous medium through capillary action.As the skilled person will appreciate, it may be possible for theaqueous medium to evaporate from the chamber. Evaporation may have animpact on the diameter of droplets dispensed from the droplet generator.The evaporation may be prevented by having a layer of a hydrophobicmedium on top of the aqueous medium. Accordingly, in some embodiments,the aqueous medium has a layer of a hydrophobic medium on top of it. Thehydrophobic medium may be any suitable hydrophobic medium. Typically,the hydrophobic medium will be a hydrophobic medium as defined herein.

There may be some applications for which only a small quantity of anaqueous medium is required. Thus, in other embodiments, the chamber isfilled with from 0.5 to 50 μl of the aqueous medium, for instance from 1to 10 μl. For instance, the chamber may be filled with about 5 μl. Inthese embodiments, the droplet generator is typically first filled withwater. The outlet of the generator may then be immersed in a wellcomprising a hydrophobic medium, which hydrophobic medium may be asherein defined. Suction may then be applied at the inlet of the dropletgenerator, for instance, by using a micropipette. By doing this, thehydrophobic medium is drawn into the outlet. For instance, the amount ofhydrophobic medium drawn into the outlet may be from 0.5 to 50 μl, forinstance from 1 to 10 μl. The outlet may then be immersed into anotherwell comprising the aqueous medium. Again, suction may be used to loadfrom 0.5 to 50 μl of the aqueous medium, for instance from 1 to 10 μl,into the outlet. The hydrophobic medium forms a plug within the nozzlethat prevents the aqueous medium in the outlet tip from mixing with thelarger volume of water. Usually, the volume of water and the hydrophobicmedium together transmit the pulse of pressure created by thepiezoelectric transducer to the tip of the outlet, where a droplet isformed from the aqueous medium. The outlet may, for instance, comprise anozzle.

When the or each droplet generator comprises a chamber for holding adroplet medium (typically an aqueous medium); an outlet; and a componentfor displacing a volume of said droplet medium through said outlet andthereby dispensing said volume as a droplet, the means for displacing avolume of said droplet medium through said outlet may be any suitablecomponent, such as any suitable moveable component, which is capable ofdisplacing a volume of the droplet medium from the chamber and throughthe outlet of the droplet generator, and which is capable of beingcontrolled by the control unit. The component for displacing a volume ofsaid droplet (e.g. aqueous) medium may be a mechanical component underthe control of the control unit. Typically, the component is apiezoelectric transducer.

Usually, the control means is adapted to communicate with the componentfor displacing a volume of said droplet (e.g. aqueous) medium throughsaid outlet, and to thereby control the dispensing of droplets of thedroplet medium from the outlet. Typically, the control means relaysinstructions to a microcontroller that is itself able to communicatewith the component. The control means is typically in electricalconnection with the microcontroller. Alternatively, the control meansmay be capable of communicating with the microcontroller wirelessly.When the component is a piezoelectric transducer, the microcontroller isable to control the application of a voltage pulse to the piezoelectrictransducer, to cause movement of the piezoelectric transducer, which inturn causes displacement of a volume of droplet medium (e.g. aqueousmedium) through the outlet of the droplet generator.

In some embodiments, the component for displacing a volume of saiddroplet medium (e.g. aqueous medium) through said outlet is apiezoelectric transducer. In other embodiments, the droplet is formedwhen a bubble of vapour behind the inlet is formed and pushed the fluidout.

Typically, the outlet has a diameter of less than 500 μm, for instance,of less than 250 μm. The diameter of the outlet provides a measure ofthe area through which a droplet is dispensed. The diameter is theinternal diameter discussed in the Supplementary Methods and isindicated in FIG. 3. When the outlet is circular, the diameter of theoutlet is the diameter of the circle. When the outlet is other thancircular, the diameter of the outlet is the diameter of a circle withthe same area as the outlet.

More typically, the outlet has a diameter of less than 200 μm, forinstance, of less than 150 μm. For instance, the outlet typically has adiameter of from 20 μm to 200 μm, for instance from 60 μm to 120 μm. Theoutlet may, for instance, have a diameter of about 100 μm.

The inventors have found that it is preferable that the outlet be assmall as possible relative to the droplets. This can help to reduce thedrag force produced when the container is moved relative to the at leastone droplet generator, for example, when the droplet assembly isdisposed in a hydrophobic medium.

Typically, the outlet is cylindrical in shape.

Usually, the or each droplet generator further comprises a capillarytube to the chamber, wherein the capillary is the nozzle and the tip ofthe capillary is said outlet. The tip of the capillary typically has adiameter of less than 150 μm. For instance the tip of the capillary mayhave a diameter of from 20 μm to 200 μm, for instance from 60 μm to 120μm. The tip of the capillary may, for instance, have a diameter of about100 μm.

Typically, the droplet generator is adapted to dispense droplets havinga diameter equal to or less than 1 mm, for instance, equal to or lessthan 200 μm.

Usually, the droplet generator is adapted to dispense droplets having adiameter of from 10 μm to 1 mm, for instance, from 10 μm to 200 μm. Thedroplet generator may, for instance, be adapted to dispense dropletshaving a diameter of from 30 μm to 60 μm. In some embodiments, the oreach droplet generator is adapted to dispense droplets having a diameterof about 50 μm.

As mentioned above, the droplets are typically dispensed into a bulkmedium. When the droplet medium is an aqueous medium the bulk medium isa hydrophobic medium. Usually, the tip of the droplet generator isimmersed in said bulk medium. However, in some embodiments, the tip ofthe droplet generator is above said bulk medium.

The inventors have found that the height between the level of thedroplet medium (usually the aqueous medium) in the chamber of the oreach droplet generator and the level of the bulk medium (usually thehydrophobic medium) may affect the diameter of the droplets dispensed.Typically, the greater the difference in height, the larger the diameterof droplets dispensed. Usually, the difference in height is from 1 cm to2 cm, for instance, about 1.5 cm.

The voltage pulse is discussed above. The required voltage pulse maydepend upon the concentration of amphipathic molecules in thehydrophobic medium. As the concentration of amphipathic moleculesincreases, the adsorption of amphipathic molecules at the interfacebetween the aqueous medium and the hydrophobic medium decreases thetension of that interface, lowering the energy required to form adroplet. Typically, the voltage pulse has a peak-to-peak amplitude offrom 5 V to 100 V, for instance, of from 10 V to 80 V. The peak-to-peakamplitude may, for instance, be of from 20 V to 60 V.

The amphipathic molecules may be any suitable amphipathic molecule.Usually, the amphipathic molecules will be ones which are capable, whenpresent in a high enough concentration, of forming a bilayer at any oneof said interfaces. The type of amphipathic molecule that is capable offorming a bilayer may, for instance, depend on additional components ofthe contacting droplets. For example, if the droplets are disposed in ahydrophobic medium, the amphipathic molecules may be any suitableamphipathic molecules capable of forming a bilayer within a hydrophobicmedium. The type of amphipathic molecules capable of forming a bilayerwithin the hydrophobic medium would typically depend on the nature ofthe hydrophobic medium and the aqueous medium of the droplets, but awide range of amphipathic molecules are possible.

Amphipathic molecules are molecules which have both hydrophobic andhydrophilic groups. The outer layer of amphipathic molecules usuallycomprises a monolayer of amphipathic molecules on the surface of thedroplet. The monolayer is typically formed and maintained naturally bythe interaction of the hydrophilic and hydrophobic groups with theaqueous medium and the bulk medium so that the molecules align on thesurface of the droplet with the hydrophilic groups facing inwardstowards the aqueous medium and the hydrophobic groups facing outwards,for instance towards a hydrophobic medium.

The amphipathic molecules may, for instance, be non-polymericamphipathic molecules. Alternatively, the amphipathic molecules may bepolymeric amphipathic molecules.

An important class of amphipathic molecules which can be used in thedroplet assembly is lipid molecules. The lipid molecules may be any ofthe major classes of lipid, including phospholipids, fatty acids, fattyacyls, glycerolipids, glycerophospholipids, sphingolipids, sterollipids, prenol lipids, saccharolipids and polyketides. Some importantexamples include phospholipids and fatty acids, for instancephospholipids. The lipid molecules may be naturally occurring orsynthetic. Whilst the formation of a bilayer from lipid molecules hasbeen demonstrated the method is expected to be appropriate for anyamphipathic molecules.

A common class of hydrophobic group that may be present in anamphipathic molecule is a hydrocarbon group, as for instance in mostlipids. However, another suitable kind of hydrophobic group that may beemployed is a fluorocarbon group. Thus, a further important class ofamphipathic molecule is an amphipathic molecule that comprises at leastone fluorocarbon group. An example of such a molecule would be alipid-like molecule which comprises a hydrophobic fluorocarbon tail anda hydrophilic head group.

The amphipathic molecules of the droplet need not be all of the sametype. Rather, the amphipathic molecules may in some embodiments be amixture of two or more different kinds of amphipathic molecule. Anotherimportant example is that the amphipathic molecules in the respectiveouter layers of different droplets in a droplet assembly may be ofdifferent types so that, if bilayers are formed, the bilayer(s) formedbetween the different droplets may be asymmetric. In some embodiments,the lipid leaflets of two contacting droplets are different.

Typically, therefore, the amphipathic molecules comprise lipidmolecules. The lipid molecules need not be all of the same type. Thus,the amphipathic molecules may comprise a single type of lipid or amixture of two or more different types of lipid molecules. Likewise,when the droplet is in contact with another droplet, the lipidcompositions of the outer layers of the individual droplets may be thesame as or different from one another. Lipid molecules are particularlyadvantageous because lipid bilayers, or more generally bilayers ofamphipathic molecules, are models of cell membranes and the dropletassembly may therefore serve as an excellent platform for a range ofexperimental studies, including for instance as novel platforms for thefundamental study of membrane proteins, or as multi-compartmentprotocellular chassis for “bottom-up” synthetic biology.

The lipid may, for instance, be sensitive to its environments (i.e. be asmart lipid). The lipid may, for instance, be sensitive to changes ispH, light or temperature. Thus the lipid may be a pH-sensitive lipid, atemperature-sensitive lipid or a light-sensitive lipid.

The lipid may allow a membrane protein (such as a natural, engineered orsynthetic membrane protein) to act as a functional component of theminimal tissue.

Phospholipids are particularly preferred for reasons outlined above andalso because they are a major component of all cell membranes, makingdroplets comprising phospholipids particularly suitable for syntheticbiology applications, as well as for drug delivery.

Accordingly, the amphipathic molecules that form an outer layer on atleast part of the surface of the aqueous medium typically comprisephospholipid molecules. The phospholipid molecules may be the same ordifferent, i.e. the amphipathic molecules comprise a single kind ofphospholipid, or a mixture of two or more different phospholipids.Phospholipids are well known to the skilled person and many arecommercially available, from suppliers such as Avanti Polar Lipids. Thephospholipid molecules may be glycerophospholipids orphosphosphingolipids or a mixture of the two. The phospholipid moleculesmay comprise anionic phospholipids, phospholipids comprising primaryamines, choline-containing phospholipids and/or glycosphingoplipids.Usually, the amphipathic molecules comprise one or moreglycerophospholipids. As the skilled person will appreciate,glycerophospholipids include, but are not limited toglycerophospholipids having a structure as defined in the followingformula (I).

wherein:

R¹ and R², which are the same or different, are selected from C₁₀-C₂₅alkyl groups and C₁₀-C₂₅ alkylene groups;

either R³ is absent such that OR³ is O⁻, or R³ is present and is H,CH₂CH₂N(R⁴)₃ ⁺, a sugar group, or an amino acid group; and

each R⁴, which is the same or different, is independently selected fromH and unsubstituted C₁-C₄ alkyl.

Typically, when R³ is CH₂CH₂N(R⁴)₃ ⁺, each R⁴, which is the same ordifferent, is selected from H and methyl. As the skilled person willappreciate, when each and every R⁴ is methyl, the R³ group is a cholinegroup, and when each and every R⁴ is H, the R³ group is an ethanolaminegroup.

When R³ is an amino acid group it may for instance be a serine group,i.e. —CH₂CH(NH₂)(COOH). When R³ is a sugar group, it may for instance beglycerol, i.e. —CH₂CHOHCH₂OH, or for instance inositol, i.e. —CH(CHOH)₅.

Typical examples of R¹ and R² groups are C₁₀-C₂₅ alkyl groups,including, but not limited to linear C₁₀-C₂₅ alkyl groups such as, forinstance, CH₃(CH₂)₁₀—, CH₃(CH₂)₁₂—, CH₃(CH₂)₁₄—, CH₃(CH₂)₁₆—,CH₃(CH₂)₁₈—, CH₃(CH₂)₂₂— and branched C₁₀-C₂₅ alkyl groups such as forinstance —CH₂—CH(CH₃)—(CH₂)₃—CH(CH₃)—(CH₂)₃—CH(CH₃)—(CH₂)₃—CH(CH₃)₂.

Further typical examples of R¹ and R² groups are unsubstituted C₁₀-C₂₅alkylene groups, including, but not limited to, CH₃(CH₂)₅CH═CH(CH₂)₇—,CH₃(CH₂)₇CH═CH(CH₂)₇—, CH₃(CH₂)₄CH═CHCH₂CH═CH(CH₂)₇—,CH₃(CH₂)₄(CH═CHCH₂)₃CH═CH(CH₂)₃—, andCH₃CH₂CH═CHCH₂CH═CHCH₂CH═CH(CH₂)₇—.

As the skilled person will appreciate, the O⁻ group in the phosphategroup adjacent to the OR³ group may in some embodiments be protonated,or associated with a suitable cation, for instance a metal cation suchas Na⁺.

Thus, the amphipathic molecules may comprise one or moreglycerophospholipids having the structure of formula (I) as definedabove.

For instance, the amphipathic molecules may comprise any one or more ofthe following glycerophospholipids:1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG) can beemployed as the amphiphilic molecules in the droplet, or a mixture ofone or more thereof. The glycerophospholipid1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) may also be used,and is typically used in combination with a pH-sensitive lipid, forinstance a fatty acid.

Additionally or alternatively, the amphipathic molecules may comprise asteroid, which steroid comprises an alkyl side-chain. The amphipathicmolecules may, for instance, comprise cholesterol, β-sitosterol andlanosterol.

In some embodiments, the amphipathic molecules comprise derivatives ofphospholipids. For instance, the amphipathic molecules may comprise aphosphatidylcholine, such as POPC(1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) or DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), or aphosphatidylglycerol, such as POPG(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol).

Preferably, the amphipathic molecules comprise DPhPC.

The amphipathic molecules may, for instance, comprise one or more fattyacids, e.g. oleic acid. Fatty acids are of course well known to theskilled person and a wide range of these are commercially available.

The amphipathic molecules may for instance comprise a mixturecomprising: (a) one or more phospholipids, and (b) one or more fattyacids.

In addition to the amphipathic molecules, the droplet may furthercomprise a PEGylated lipid. The term “PEGylated lipid”, as used herein,refers to a lipid which has been derivatised with poly(ethylene glycol).

A PEGylated lipid may be particularly useful when the droplet assemblyis in a volume (such as a drop) of a hydrophobic medium, which volume isin a hydrophilic medium such as an aqueous medium. A PEGylated lipidmay, for example, be particularly useful when the droplet assembly formspart of a droplet encapsulate (for example, as defined below). Theencapsulate may, for instance, be functionalised using a PEGylatedlipid.

The inclusion of one or more PEGylated lipids in the droplet typicallystabilises the droplet assembly in vivo, and in particular prolongs theplasma half-life of the droplet assembly. This means that, when thedroplet assembly contains one or more therapeutic or diagnostic agents,for instance if it is being used as a drug-delivery vehicle theinclusion of one or more PEGylated lipids may also have the usefuleffect of prolonging the plasma half-life of the agent within a dropletassembly. Such effects have been observed previously when PEGylatedlipids are used in liposomal drug formulations. PEGylated lipids areknown in the art and are commercially available from suppliers such asNOF Corporation, Japan (seehttp://www.phospholipid.jp/phospholipid_2-3.html). Any suitablePEGylated lipid may be employed, including, but not limited to,PEG-phospholipids, diacylglycerol-PEG, cholesterol-PEG derivatives, andmixtures thereof.

The poly(ethylene glycol) (PEG) component of the PEGylated lipid mayhave any one of several different geometries. Thus, it could besubstantially linear PEG or branched PEG. The branched PEG may forinstance have from three to ten PEG chains emanating from a central coregroup. Alternatively, the branched PEG could be a star PEG, having from10 to 100 PEG chains emanating from a central core group. Alternatively,the PEG may be a comb PEG, having multiple PEG chains grafted to apolymer backbone.

The one or more PEGylated lipids employed may for instance comprise aPEG-phospholipid of the following formula (II)

wherein R¹ and R² are as defined above for the glycerophospholipids offormula (I), and R⁵ is a group which comprises poly(ethylene glycol).

The group which comprises poly(ethylene glycol) may for instance havethe formula —CH₂CH₂NHC(O)—X, or for instance —CH₂CH₂NHC(O)(CH₂)₃C(O)—Xwherein X comprises said poly(ethylene glycol). The group X may forinstance comprise substantially linear PEG, or for instance a branchedPEG, having, for instance, from three to ten PEG chains emanating from acentral core group. Alternatively, it can be a star PEG, having, forinstance, from 10 to 100 PEG chains emanating from a central core group.Or for instance it may be a comb PEG, having multiple PEG chains graftedto a polymer backbone.

Thus, R⁵ may for instance be —CH₂CH₂NHC(O)—(OCH₂CH₂)_(q)OCH₃,—CH₂CH₂NHC(O)(CH₂)₃C(O)—(OCH₂CH₂)_(q)OCH₃,—CH₂CH₂NHC(O)—(OCH₂CH₂)_(q)OH, or—CH₂CH₂NHC(O)(CH₂)₃C(O)—(OCH₂CH₂)_(q)OH, wherein q is a positiveinteger. The integer q may for instance be from 5 to 10,000, or forinstance from 10 to 1,000.

Alternatively, R⁵ may be —(CH₂CH₂O)_(q)CH₃ or —(CH₂CH₂O)_(q)H, wherein qis a positive integer. The integer q may for instance be from 5 to10,000, or for instance from 10 to 1,000.

Additionally or alternatively, the one or more PEGylated lipids maycomprise a diacylglycerol-PEG of formula (III)

wherein R¹ and R² are as defined above for the glycerophospholipids offormula (I), and R⁶ is a group which comprises poly(ethylene glycol).

The poly(ethylene glycol) may for instance comprise substantially linearPEG, or for instance a branched PEG, having, for instance, from three toten PEG chains emanating from a central core group. Alternatively, itcan be a star PEG, having, for instance, from 10 to 100 PEG chainsemanating from a central core group. Or for instance it may be a combPEG, having multiple PEG chains grafted to a polymer backbone.

R⁶ may for instance be —(CH₂CH₂O)_(q)CH₃, —(CH₂CH₂O)_(q)H,—CH₂CH₂NHC(O)—(OCH₂CH₂)_(q)OCH₃, —CH₂CH₂NHC(O)—(OCH₂CH₂)_(q)OH,—CH₂CH₂NHC(O)(CH₂)₃C(O)—(OCH₂CH₂)_(q)OCH₃ or—CH₂CH₂NHC(O)(CH₂)₃C(O)—(OCH₂CH₂)_(q)OH wherein q is a positive integer.The integer q may for instance be from 5 to 10,000, or for instance from10 to 1,000.

Additionally or alternatively, the one or more PEGylated lipids maycomprise a cholesterol-PEG derivative of formula (IV)

wherein R⁷ is a group which comprises poly(ethylene glycol).

Again, the poly(ethylene glycol) may comprise substantially linear PEG,or for instance a branched PEG, having, for instance, from three to tenPEG chains emanating from a central core group. Alternatively, it can bea star PEG, having, for instance, from 10 to 100 PEG chains emanatingfrom a central core group. Or for instance it may be a comb PEG, havingmultiple PEG chains grafted to a polymer backbone.

R⁷ may for instance be —(OCH₂CH₂)_(q)OH or —(OCH₂CH₂)_(q)OCH₃ wherein qis a positive integer. The integer q may for instance be from 5 to10,000, or for instance from 10 to 1,000.

Polyglycerine may be used instead of poly(ethylene glycol).

The concentration of amphipathic molecules may be any suitableconcentration.

Typically, the concentration of amphipathic molecules is less than orequal to 15 mg mL⁻¹. For instance, the concentration of amphipathicmolecules may be from 0 to 10 mg mL⁻¹. Usually, the concentration ofamphipathic molecules is from 0.05 mg mL⁻¹ to 10 mg mL⁻¹, for instance,from 0.05 mg mL⁻¹ to 5 mg mL⁻¹. More typically, the concentration ofamphipathic molecules is from 0.1 mg mL⁻¹ to 2.5 mg mL⁻¹, for instance,from 0.2 mg mL⁻¹ to 0.5 mg mL⁻¹.

Typically, the droplet medium is an aqueous medium and the droplet isdisposed in a hydrophobic medium and the concentration of amphipathicmolecules is the concentration of amphipathic molecules in thehydrophobic medium.

Additionally or alternatively, when the droplets are formed, the aqueousmedium of the droplets may comprise amphipathic molecules. Theconcentration of amphipathic molecules may therefore be theconcentration of amphipathic molecules in the aqueous medium.

The container of the apparatus of the invention usually contains a bulkmedium. When the droplet medium is an aqueous medium, the bulk medium isa hydrophobic medium. When the droplet medium is a hydrophobic medium,the bulk medium is an aqueous medium.

Typically, the droplet medium is an aqueous medium and the container ofthe apparatus of the invention contains a hydrophobic medium.

The hydrophobic medium may be selected from a wide range of materials.The hydrophobic medium may comprise a single hydrophobic compound.Alternatively, it may comprise a mixture of two or more differenthydrophobic compounds. The hydrophobic medium can, for instance, beselected to affect the buoyancy of the droplet and the speed offormation of the layer of amphipathic molecules around at least part ofthe droplet after the droplet is first introduced into the hydrophobicmedium.

The hydrophobic medium is typically an oil. The oil may be a single,pure, compound, or the oil may comprise a mixture of two or morecompounds. It is usually desirable that the oil does not significantlydestabilize any bilayers formed.

The oil may for instance comprise silicone oil (for instance poly phenylmethyl siloxane). The oil may consist of a single silicone oil, forinstance poly phenyl methyl siloxane. Alternatively, the oil maycomprise a mixture of two or more different silicone oils.

Any suitable silicone oil may be used. For instance, the oil maycomprise silicon oil DC200 (a polymer comprising monomer units of—O—Si(CH₃)₂—), poly(dimethylsiloxane) (PDMS), hydroxy terminated, orPDMS 200. In some embodiments, the silicone oil is apoly(methylphenylsiloxane), such as AR20.

Additionally or alternatively, the oil may comprise a hydrocarbon. Whenthe oil comprises a hydrocarbon it may comprise a single hydrocarboncompound, or a mixture of two or more hydrocarbons.

In some embodiments, the oil is a mixture comprising: (a) one or morehydrocarbons, and (b) one or more silicone oils. The hydrocarbon may,for instance, be any suitable liquid hydrocarbon. Whether a particularhydrocarbon is liquid will depend upon the temperature of thehydrophobic medium. Thus the term liquid hydrocarbon refers to ahydrocarbon that is a liquid at the temperature that the hydrophobicmedium is at. Typically, the hydrophobic medium will be at roomtemperature. However, in some embodiments, the hydrophobic medium may beabove or below room temperature.

In some embodiments, the oil may comprise a solid. A solid hydrocarbonmay, for instance, be used in combination with a silicone oil. The oilmay, for instance, be a mixture of solids that dissolve to form aliquid.

When the oil comprises a hydrocarbon, the hydrocarbon may be branched orunbranched, for example a hydrocarbon having from 5 to 40 carbon atoms,or from 5 to 30 carbon atoms (although hydrocarbons of lower molecularweight would require control of evaporation). Preferably, thehydrocarbon is a liquid at the operating temperature of the droplet usedin the invention. Suitable examples include alkanes or alkenes, such ashexadecane, decane, pentane or squalene. Usually, the oil comprises ahydrocarbon.

Typically the hydrocarbon is an unsubstituted C₁₀-C₂₀ alkane, forinstance hexadecane.

Shorter alkanes may be suitable, for instance, in assemblies for whichbuoyancy effects are less important and whose outer layer of amphipathicmolecules, on at least part of the surface of the droplet, may form morequickly.

In some embodiments the hydrocarbon is a longer-chain hydrocarbon, suchas unsubstituted C₁₅-C₄₀ alkane. For instance, an unsubstituted C₁₆-C₃₀alkane chain, such as squalene.

In one embodiment, the hydrophobic medium comprises an unsubstitutedC₁₀-C₂₀ alkane and the amphipathic molecules comprise one or moreglycerophospholipids. For instance, the hydrophobic medium may comprisehexadecane and the outer layer of amphipathic molecules may compriseDPhPC.

Other types of oil are possible. For example the oil may be afluorocarbon. This might be useful for the study of some systems, forexample to minimise loss of a particular membrane protein or solute fromthe droplet assembly or to control the content of gases such as oxygen.Because fluorocarbons can be both hydrophobic and lipophobic, an oilphase that comprises fluorocarbons can usefully prevent the adhesion ofa droplet assembly to surfaces.

In another embodiment, the hydrocarbon is a bromo-substituted C₁₀-C₃₀alkane, or for instance a bromo-substituted C₁₀-C₂₀ alkane, e.g.bromododecane.

Typically, the oil comprises silicone oil or a hydrocarbon. Any suitablesilicone oil may be employed. Usually, the silicone oil is as hereindefined.

Silicone oil is advantageous on account of its density being close tothat of water, which ensures that the droplet is approximately neutrallybuoyant in water. The silicone oil may for instance be poly phenylmethyl siloxane, which has a density of about 1 g cm⁻³.

The hydrocarbon typically has from 5 to 40 carbon atoms (a C₅-C₄₀hydrocarbon), more typically from 10 to 30 carbon atoms (a C₁₀-C₃₀hydrocarbon). Typically, it is an alkane or an alkene. Thus, thehydrocarbon may be a C₅-C₃₀ alkane, or a C₁₀-C₂₀ alkane. In anotherembodiment, the hydrocarbon may be a C₅-C₂₀ alkene, or a C₁₀-C₂₀ alkene.The hydrocarbon is typically unsubstituted. In one embodiment it issqualene. In a preferred embodiment, the hydrocarbon is an unsubstitutedC₅-C₂₀ alkane, preferably an unsubstituted C₁₀-C₂₀ alkane.

The hydrocarbon may for instance be squalene, hexadecane or decane.However, in some embodiments the hydrocarbon may be substituted with ahalogen atom, for instance bromine.

In some embodiments, the hydrophobic medium comprises a mixture ofsilicone oil and a hydrocarbon. Such mixtures have been found to provideadvantageously short incubation times required for stable bilayers to beformed. The silicone oil and hydrocarbon in the mixture may be asfurther defined above. Typically, the hydrocarbon is an unsubstitutedC₁₀-C₂₀ alkane, preferably hexadecane. The silicone oil usually has adensity close to, but less than, that of water, to control the sinkingrate of droplets during printing. When the droplet assembly is in avolume (such as a drop) of a hydrophobic medium, which volume is in ahydrophilic medium (such as an aqueous medium), the silicone oiltypically has a density close to that of water to ensure the droplets inthe droplet assembly have approximately neutral buoyancy in thehydrophilic medium.

The silicon oil may for instance be poly phenyl methyl siloxane.Usually, the volume ratio of the silicone oil to the hydrocarbon isequal or greater than 0.5:1. The volume ratio of the silicone oil to thehydrocarbon may for instance be from 0.5:1 to 5:1, for instance about1:1. In some embodiments, the volume ratio of the silicone oil to thehydrocarbon is equal to or greater than 5:1.

The hydrophobic medium employed may, for instance, have a density closeto that of water, for instance a density of less than or equal to about1 g cm⁻³.

In one embodiment, the hydrophobic medium comprises both silicone oiland hexadecane. Typically the silicone oil is poly phenyl methylsiloxane. The volume ratio of the silicone oil to the hexadecane istypically equal or greater than 0.5:1, for instance from 0.5:1 to 5:1.It may for instance be about 1:1. In some embodiments, the volume ratioof the silicone oil to the hydrocarbon is equal to or greater than 5:1.

Preferably, the hydrophobic medium comprises hexadecane. In someembodiments, the hydrophobic medium further comprises silicone oil.

Typically, the hydrophobic medium comprises hexadecane and theamphipathic molecules comprise DPhPC. More typically, the hydrophobicmedium comprises hexadecane, the amphipathic molecules comprise DPhPCand the aqueous medium comprises an aqueous buffer solution.

The container of the apparatus of the invention may be any suitablecontainer.

Typically, the container comprises a polymer, such as poly(methylmethacrylate). For instance, the container may comprise a wellmicromachined from said polymer.

In some embodiments, the bottom surface of the container comprisesglass. At least one droplet of said plurality of droplets is typicallyin contact with the glass. The glass prevents the droplet from movingaround.

In other embodiments, the bottom surface of the container comprises apolymer, such as poly(methyl methacrylate). For instance, when theapparatus of the invention is used to produce a self-folding dropletassembly (for instance a self-folding assembly as discussed below), atleast one droplet of said plurality of droplets is typically in contactwith a polymer. Droplets of the self-folding droplet assembly usuallydoes not adhere to the polymer surface, allowing the droplet assembly tofold.

In a further embodiment, the container comprises a polymer such aspolystyrene. For instance, the container may comprise polystyrene whenthe apparatus of the invention is used to produce a droplet assembly inwhich the aqueous droplets are in a drop of a hydrophobic medium whichis in turn within a second bulk medium which is an aqueous medium. Suchdroplet assemblies are discussed below.

Typically, the container contains the bulk medium, which is ahydrophobic medium, and amphipathic molecules. Additionally oralternatively, the droplet generator may comprise the droplet medium,which is an aqueous medium, and amphipathic molecules.

Usually, the droplet generator in the apparatus of the inventioncontains the droplet medium. As mentioned hereinbefore, the dropletmedium is typically an aqueous medium. The aqueous medium may, forinstance, be as further defined herein. In some embodiments, the dropletgenerator contains an aqueous medium and a membrane protein. Usually,the membrane protein is in an aqueous solution. Additionally oralternatively, the bulk hydrophobic medium may comprise a membraneprotein.

The membrane protein may be of any type. The use of integral membraneproteins has been demonstrated, but it is equally expected thatperipheral membrane proteins could be used. The membrane protein may forinstance be a membrane pump, channel and/or pore, to allow for precisecontrol over the exchange of material, and electrical communication,between (i) individual droplets within the assembly and (ii) the dropletassembly and an external solution. The membrane protein could forinstance be an α-hemolysin (αHL) pore, such as a staphylococcalα-hemolysin pore. However, any suitable membrane protein can be usedincluding one from the two major classes, that is, β-barrels orα-helical bundles. An important application is a membrane protein whichis a pore or a channel. Besides a protein pore or channel, furtherpossible membrane proteins include, but not exclusively, a receptor, atransporter or a protein which effects cell recognition or acell-to-cell interaction. The channel can be a voltage-gated ionchannel, a light-sensitive channel such as bacteriorhodopsin, aligand-gated channel or a mechano-sensitive channel.

Suitable membrane proteins which allow for exchange of materials andelectrical communication are known and readily available to the skilledperson; many such proteins are either commercially available or can beprepared by known methods. For instance, wild type (WT) αHL monomers canbe prepared by in vitro transcription-translation (IVTT), andheptamerised by incubation with rabbit red blood cell membranes. Theheptamers are typically purified by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) (Maglia, G. et al.,Method. Enzymol. 475, 591-623, (2010). Also, Bayley, H. et al., Dropletinterface bilayers. Mol. BioSyst. 4, 1191-1208 (2008) lists severalproteins that were tested for insertion into droplet interface bilayersmade in bulk oil. Other suitable membrane protein include, but are notlimited to, bacterial peptides and ionophores.

The membrane protein may, alternatively, be an engineered membraneprotein or synthetic membrane protein. The engineered membrane proteinmay, for instance, be a genetically engineered protein, or a covalent ornon-covalent chemically engineering protein. The synthetic membraneprotein may, for instance, be a peptide or an organic molecule.

Droplets can exchange chemical species with each other through membraneproteins incorporated in the bilayer between the droplets. Suitablemembrane proteins include, but are not limited to, pumps, channelsand/or pores, receptor proteins, transporter proteins, and/or proteinswhich effect cell recognition or a cell-to-cell interaction, forinstance an αHL pore. Other suitable membrane protein include, but arenot limited to, bacterial peptides and ionophores. The membrane proteinmay, alternatively, be an engineered membrane protein or syntheticmembrane protein.

Thus, a droplet assembly may be capable of trafficking materials such aschemical compounds through the network, from object to object, as wellas to and from the external environment. A droplet assembly may,additionally or alternatively, be capable of transferring electricalsignals from object to object, or to and from the external environment.Complex transport systems can be built up in this way. The transportsystem comprises a droplet assembly.

Chemical species such as water may be exchanged through the bilayer.

A droplet assembly may, for instance, act as a sensor module, capable ofsensing the presence of a particular chemical in the externalenvironment, for instance, or capable of sensing light. Thus, a dropletmay comprise a sensor molecule. The sensor molecule can be present inthe aqueous medium of the droplet or in a bilayer and may be anysuitable sensor molecule. The sensor molecule may be a molecule which issensitive to the presence of a particular chemical (for instance atarget analyte), or it may be a light-sensitive molecule, or it may besensitive to changes in pH or temperature. In some embodiments, thesensor molecule may be sensitive to the presence (or absence) of aparticular biochemical or enzyme.

Typically, the membrane protein is α-hemolysin (αHL).

Usually, when present, the concentration of membrane proteins is equalto or greater than 0.1 ng mL⁻¹, for instance, equal to or greater than 1ng mL⁻¹. For instance, the concentration of membrane proteins is equalto or greater than 2 ng mL⁻¹, for instance, equal to or greater than 10ng mL⁻¹. The concentration of membrane proteins may, for instance, befrom 0.1 ng mL⁻¹ to 100 mg mL⁻¹. Typically, the concentration ofmembrane proteins is from 0.1 ng mL⁻¹ to 10 mg mL⁻¹, for instance from 1ng mL⁻¹ to 5 mg mL⁻¹. More typically, the concentration of membraneproteins is from 2 ng mL⁻¹ to 1 mg mL⁻¹, for instance, from 2 ng mL⁻¹ to0.1 mg mL⁻¹. In some embodiments, the concentration of membrane proteinsis about 10 ng mL⁻¹.

Typically, the concentration of membrane proteins is the concentrationof membrane proteins in the aqueous medium of the droplet, when thedroplet is formed. When a droplet comprising a membrane protein iscontacted with another droplet (which may or may not comprise a membraneprotein), a bilayer of amphipathic molecules is formed at the interface.That bilayer typically comprises a membrane protein. Therefore membraneproteins initially in the aqueous medium may move to the bilayer.

Suitable concentrations of the membrane protein may depend on a numberof factors. The rate of insertion of the membrane protein into thebilayer may, for instance, decrease with time. Typically, this will puta lower limit on the concentrations of the membrane protein that may beused.

Surfactants may be added to the aqueous medium. The surfactant may, forinstance, be added to catalyse the movement of membrane proteins fromthe aqueous medium into the bilayer. Usually, the concentration of thesurfactant added would not be high enough to destabilize the bilayers.

The concentration may also depend on the time it takes to print thedroplet assembly. If, for instance, the number of droplets in thedroplet assembly is reduced, the concentration of the membrane proteinmay be reduced also.

When the droplet is in contact with another droplet, the concentrationof membrane proteins in the droplet and the other droplet may be thesame or different. Further, when the droplet is part of a dropletassembly, the concentration of membrane proteins in each droplet of thedroplet assembly may be the same or different.

Typically, at least one bilayer comprises a membrane protein. Thebilayer at an interface between contacting droplets, may comprise morethan one membrane protein. For instance, a particular bilayer maycontain multiple copies of the same membrane protein, or two or moredifferent classes of membrane proteins. Where more than one class ispresent, the bilayer may contain multiple copies of each differentclass.

Suitable membrane proteins which allow for exchange of materials andelectrical communication are known and readily available to the skilledperson; many such proteins are either commercially available or can beprepared by known methods. For instance, WT αHL monomers can be preparedby in vitro transcription-translation (IVTT), and heptamerised byincubation with rabbit red blood cell membranes. The heptamers aretypically purified by sodium dodecyl sulphate polyacrylamide gelelectrophoresis (SDS-PAGE) (Maglia, G. et al., Method. Enzymol. 475,591-623, 2010). Also, Bayley, H. et al., Droplet interface bilayers.Mol. BioSyst. 4, 1191-1208 (2008) lists several proteins that weretested for insertion into droplet interface bilayers made in bulk oil.Other suitable membrane protein include, but are not limited to,bacterial peptides and ionophores.

The membrane protein may, alternatively, be an engineered membraneprotein or synthetic membrane protein. The engineered membrane proteinmay, for instance, be a genetically engineered protein, or a covalent ornon-covalent chemically engineering protein. The synthetic membraneprotein may, for instance, be a peptide or an organic molecule.

A droplet may, in some embodiments, comprise other materials, compoundsor substances. For instance, a droplet may comprise a small molecule,such as a dye, or a magnet. Suitable dyes include, but are not limitedto, xylene cyanol FF, orange G, pyranine, fluorescein and 5-cTAMRA(5-carboxytetramethylrhodamine). Alternatively, a droplet may compriseany suitable sensor molecule, for instance a sensor molecule that itsensitive to a particular chemical or is a light-sensitive molecule, orit may be sensitive to changes in pH or temperature. In someembodiments, the sensor molecule may be sensitive to the presence (orabsence) of a particular biochemical or enzyme. As a furtheralternative, a droplet may comprise a therapeutic agent, such as aprodrug, or a diagnostic agent, such as a contrast agent. A droplet maycomprise an enzyme.

A droplet may comprise a living cell (or living cells), for example foruse in tissue engineering. For instance, the cells may be allowed togrow inside the droplet after printing and/or to break down the bilayersbetween droplets after printing.

A droplet assembly, once printed, may be used as a template for thepatterning of a solid material. The solid material may, for instance, beused in electronics, optics, photonics, or other material scienceapplications. A droplet, or droplets, may, for instance, compriseinorganic materials that could diffuse between specific droplets. Theinorganic materials may then react to form inorganic solids such ascadmium sulphide.

In the apparatus of the invention, the or each droplet generator istypically adapted to dispense droplets having a diameter of equal to orless than 1 mm, for instance, equal to or less than 200 μm.

Typically, the droplets are spherical droplets and the diameter is equalto the diameter of a sphere. When the droplets are not sphericaldroplets, the diameter of the droplet is equal to the diameter of asphere that has the same volume as the droplet.

As discussed above, the droplet size may be controlled by a variety offactors such as the nozzle geometry, the voltage pulse amplitude and theduration of the voltage pulse.

Usually, the or each droplet generator is adapted to dispense dropletshaving a diameter of from 10 μm to 1 mm. The or each droplet generatormay, for example, be adapted to dispense droplets having a diameter offrom 10 μm to 200 μm, for instance, of from 30 μm to 60 μm. In someembodiments, the or each droplet generator is adapted to dispensedroplets having a diameter of about 50 μm.

Typically, the or each droplet generator is adapted to dispense dropletshaving a volume of equal to or less than 2 μL.

In some embodiments, the or each droplet generator is adapted todispense droplets having a volume of from 0.001 nL to 100 nL, forinstance, from 0.005 nL to 0.5 nL.

Typically, the control unit is adapted to control the dispensing ofdroplets at a rate of at least 0.01 s⁻¹, for instance, 0.25 s⁻¹.Usually, the control unit is adapted to control the dispensing ofdroplets at a rate of at least 0.5 s⁻¹.

Usually, the control unit is adapted to control the dispensing ofdroplets at a rate of from 0.01 to 100 s⁻¹, for instance, from 0.01 to50 s⁻¹. Typically, the control unit is adapted to control the dispensingof droplets at a rate of from 0.01 to 10 s⁻¹, for instance, from 0.25 to5 s⁻¹. More typically, the control unit is adapted to control thedispensing of droplets at a rate of from 0.5 to 2.5 s⁻¹, for instance,from 0.75 to 2 s⁻¹. Usually, the control unit is adapted to control thedispensing of droplets at a rate of about 1 s⁻¹.

When there are two or more droplet generators, the rate at whichdroplets are dispensed is the rate at which droplets are dispensed froman individual droplet generator and not necessarily the overall rate atwhich droplets are dispensed. For example, if multiple dropletgenerators are used in parallel, the overall printing rate wouldtypically be approximately equal to the rate at which droplets aredispensed from one droplet generator, multiplied by the number ofgenerators.

The use of more than one droplet generator may thus significantlyincrease the overall printing rate.

In some embodiments, there may be a pause between the dispensing of onedroplet and the dispensing of another droplet. The dispensing ofdroplets is discussed in more detail below, for the process of theinvention. The printing patterns and algorithms used are alsoillustrated in the Examples, under Supplementary Methods.

Usually, the apparatus of the invention comprises a plurality of saiddroplet generators. For instance, the apparatus may comprise two or moredroplet generators. Typically, the apparatus comprises from 1 to 20droplet generators, for instance from 1 to 10 droplet generators. Moretypically, the apparatus comprises from 1 to 5 droplet generators, forinstance, 2 or 3 droplet generators. In some embodiments, the apparatuscomprises two droplet generators.

Typically, the control unit is adapted to control the dispensing ofdroplets from each droplet generator.

As discussed above, an advantage of the apparatus comprising a pluralityof said droplet generators is that each droplet generator may comprise adifferent droplet medium (typically aqueous medium). This allows diversedroplet assemblies to be printed. The droplet assemblies may, forinstance, comprise multiple compartments with different functionalitiesand/or complex communications systems that allow droplets within theassemblies to communicate with each other or with their externalenvironment.

Accordingly, in some embodiments, when the apparatus comprises aplurality of said droplet generators, the apparatus usually comprises afirst droplet generator comprising a first droplet medium and a seconddroplet generator comprising a second droplet medium, wherein the firstand second droplet media are different. The first droplet medium isusually an aqueous medium as herein defined. The second droplet mediumis typically also an aqueous medium as herein defined. Accordingly, insome embodiments, when the apparatus comprises a plurality of saiddroplet generators, the apparatus comprises a first droplet generatorcomprising a first aqueous medium and a second droplet generatorcomprising a second aqueous medium, wherein the first and second aqueousmedia are different. The first aqueous medium is usually an aqueousmedium as herein defined. The second aqueous medium is typically anaqueous medium as herein defined.

In some embodiments, the first aqueous medium comprises a membraneprotein and the second aqueous medium does not comprise said membraneprotein. The membrane protein may, for instance, be a membrane proteinas herein defined. The membrane protein may for instance be a membranepump, channel and/or pore, to allow for precise control over theexchange of material, and electrical communication, between (i)individual droplets within the assembly and (ii) the droplet assemblyand an external solution. The membrane protein could for instance be anαHL pore. However, any suitable membrane protein can be used includingone from the two major classes, that is, β-barrels or α-helical bundles.Besides a protein pore or channel, further possible membrane proteinsinclude, but not exclusively, a receptor, a transporter or a proteinwhich effects cell recognition or a cell-to-cell interaction. Thechannel can be a voltage-gated ion channel, a light-sensitive channelsuch as bacteriorhodopsin, a ligand-gated channel or a mechano-sensitivechannel. In some embodiments, the membrane protein is in an aqueoussolution. Other suitable membrane protein include, but are not limitedto, bacterial peptides and ionophores. The membrane protein may,alternatively, be an engineered membrane protein or synthetic membraneprotein. The engineered membrane protein may, for instance, be agenetically engineered protein, or a covalent or non-covalent chemicallyengineering protein. The synthetic membrane protein may, for instance,be a peptide or an organic molecule.

In another embodiment, the first and second aqueous media may havedifferent concentrations of a membrane protein. The membrane protein mayfor instance be a membrane pump, channel and/or pore, to allow forprecise control over the exchange of material, and electricalcommunication, between (i) individual droplets within the assembly and(ii) the droplet assembly and an external solution. The membrane proteincould for instance be an αHL pore. However, any suitable membraneprotein can be used including the two major classes, that is, β-barrelsor α-helical bundles. Besides a protein pore or channel, furtherpossible membrane proteins include, but not exclusively, a receptor, atransporter or a protein which effects cell recognition or acell-to-cell interaction. The channel can be a voltage-gated ionchannel, a light-sensitive channel such as bacteriorhodopsin, aligand-gated channel or a mechano-sensitive channel. Other suitablemembrane protein include, but are not limited to, bacterial peptides andionophores. The membrane protein may, alternatively, be an engineeredmembrane protein or synthetic membrane protein. The engineered membraneprotein may, for instance, be a genetically engineered protein, or acovalent or non-covalent chemically engineering protein. The syntheticmembrane protein may, for instance, be a peptide or an organic molecule.

In some embodiments, the first and second aqueous media have differentosmolarities, or the first and second aqueous media comprise differentconcentrations of a salt or buffer. For instance, when the first aqueousmedium comprises a membrane protein, the first and second aqueous mediamay have different osmolarities, or the first and second aqueous mediamay comprise different concentrations of a salt or buffer. In otherembodiments, the first aqueous medium comprises a first salt and thesecond aqueous medium comprises a second salt, wherein the first saltand the second salt are different. The first salt may, for instance, bea chloride, such as potassium chloride, and the second salt may be acarbonate, such as potassium carbonate.

The ratio of the osmolarity of the first aqueous medium to theosmolarity of the second aqueous medium may be from 2:1 to 50:1, and ispreferably from 5:1 to 20:1. In some embodiments, the ratio of theosmolarity of the first aqueous medium to the osmolarity of the secondaqueous medium is from 5:1 to 15:1.

The ratio of the concentration of the salt or buffer in the firstaqueous medium to the concentration of the salt or buffer in the secondaqueous medium may be from 2:1 to 50:1, and is preferably from 5:1 to20:1.

In some embodiments, the concentration of the salt or buffer in thefirst aqueous medium is from 100 mM to 1,000 mM and the concentration ofthe salt or buffer in the second aqueous medium is from 0.1 mM to 100mM. Typically, the concentration of the salt or buffer in the firstaqueous medium is from 150 mM to 750 mM and the concentration of thesalt or buffer in the second aqueous medium is from 0.5 mM to 75 mM.More typically, the concentration of the salt or buffer in the firstaqueous medium is from 150 mM to 500 mM and the concentration of thesalt or buffer in the second aqueous medium is from 5 mM to 25 mM. Forinstance, the concentration of the salt or buffer in the first aqueousmedium may be about 250 mM and the concentration of the salt or bufferin the second aqueous medium be about 25 mM.

Usually, the concentration of the salt or buffer in the first aqueousmedium and the concentration of the salt or buffer in the second aqueousmedium is a concentration of an alkali metal halide salt, such aspotassium chloride. For instance, the buffer solution may compriseTris-HCl and/or KCl.

When two droplets comprising different concentrations of a salt areplaced in contact with each other, water will typically transfer fromone droplet to the other. The inventors have used this process to theiradvantage by creating networks of droplets of different osmolarities.The transfer of water between the droplets causes deformation of thenetwork as long as adhesion between droplets is maintained, asillustrated in FIGS. 15A-15E. By controlling factors such as thedifference in osmolarity of droplets in the network, the apparatus ofthe invention may be adapted to produce a droplet assembly that folds ina predictable way, referred to herein as a self-folding network orself-folding droplet assembly. The apparatus may, for instance, beadapted to produce a droplet assembly as defined herein below in theproduct of the invention.

When osmosis occurs between contacting droplets in the droplet assemblythat comprise different aqueous media, the flow of water from onedroplet to another will cause a change in the relative size of the twodroplets. Therefore, two droplets may, for instance, have the samediameter when they first come into contact with each other but they mayhave different diameters after osmosis has taken place.

In the apparatus of the invention, a first droplet generator may beadapted to dispense droplets having a first diameter, and a seconddroplet generator may be adapted to dispense droplets having a seconddiameter, wherein the first and second diameters are the same ordifferent. In some embodiments, the first and second diameters are thesame, when the droplets first come into contact with each other. Inother embodiments, the first and second diameters are the different,when the droplets first come into contact with each other.

As mentioned above, the or each droplet generator typically comprises achamber for holding a droplet medium (typically an aqueous medium); anoutlet; and a component for displacing a volume of said droplet mediumthrough said outlet and thereby dispensing said volume as a droplet.When, the apparatus comprises a plurality of said droplet generators,the outlet of the first droplet generator may have the same diameter asthe outlet of the second droplet generator or it may have a differentdiameter from the outlet of the second droplet generator.

In some embodiments, the control unit is adapted to coordinate (a) themovement of the container relative to the or each droplet generator and(b) the dispensing of the droplets, to create a said droplet assemblywhich comprises at least one layer of droplets, wherein each of saiddroplets comprises (i) a droplet medium and (ii) an outer layer ofamphipathic molecules around the surface of the droplet medium, whereinthe droplet medium is an aqueous medium or a hydrophobic medium andwherein each droplet in the layer contacts at least one other droplet inthe layer to form a layer of said amphipathic molecules as an interfacebetween contacting droplets.

The layer of said amphipathic molecules which is an interface betweencontacting droplets may, for instance, be a bilayer of said amphipathicmolecules or a layer of a block copolymer. The layer of a blockcopolymer may, for example, be a layer of a triblock copolymer. When thedroplet medium is an aqueous medium the layer of said amphipathicmolecules which is an interface between contacting droplets may, forinstance, be a bilayer of said amphipathic molecules or a layer of ablock copolymer. When the droplet medium is a hydrophobic medium thelayer of said amphipathic molecules which is an interface betweencontacting droplets may, for instance, be a bilayer of said amphipathicmolecules or a layer of a block copolymer.

Usually, the layer of said amphipathic molecules which is an interfacebetween contacting droplets is a bilayer of said amphipathic molecules.

Typically the droplet medium is an aqueous medium. Thus, in someembodiments, the control unit is adapted to coordinate (a) the movementof the container relative to the or each droplet generator and (b) thedispensing of the droplets, to create a said droplet assembly whichcomprises at least one layer of droplets, wherein each of said dropletscomprises (i) an aqueous medium and (ii) an outer layer of amphipathicmolecules around the surface of the aqueous medium, and wherein eachdroplet in the layer contacts at least one other droplet in the layer toform a bilayer of said amphipathic molecules as an interface betweencontacting droplets.

The aqueous medium is usually as defined herein. The aqueous medium may,for instance, be any suitable aqueous medium. For instance, the aqueousmedium may be pure water, or an aqueous buffer solution, or an aqueoussolution of one or more salts, or an aqueous solution comprising agaroseand water.

The amphipathic molecules may be as further defined herein.

Usually, the control unit is adapted to coordinate (a) the movement ofthe container relative to the or each droplet generator and (b) thedispensing of the droplets, to create a said droplet assembly whichcomprises a plurality of said layers of said droplets, wherein eachlayer is disposed adjacent to another layer, so that droplets in a layercontact droplets in an adjacent layer to form layers of amphipathicmolecules as interfaces between the contacting droplets.

The layer of said amphipathic molecules which is an interface betweencontacting droplets may, for instance, be a bilayer of said amphipathicmolecules or a layer of a block copolymer. The layer of a blockcopolymer may, for example, be a layer of a triblock copolymer. When thedroplet medium is an aqueous medium the layer of said amphipathicmolecules which is an interface between contacting droplets may, forinstance, be a bilayer of said amphipathic molecules or a layer of ablock copolymer. When the droplet medium is a hydrophobic medium thelayer of said amphipathic molecules which is an interface betweencontacting droplets may, for instance, be a bilayer of said amphipathicmolecules or a layer of a block copolymer.

Typically, the layer of said amphipathic molecules which is an interfacebetween contacting droplets is a bilayer of said amphipathic molecules.

Usually, the control unit is adapted to coordinate (a) the movement ofthe container relative to the or each droplet generator and (b) thedispensing of the droplets, to create a said droplet assembly whichcomprises a plurality of said layers of said droplets, wherein one layeris deposited on another layer. Typically, the droplet assembly is builtupwards in horizontal layers.

The droplet assembly may, for instance, comprise three or more layers.The number of layers of said droplets may, for instance, be equal to orgreater than 5. Typically, the number of layers of said droplets isequal to or greater than 10, for instance, equal to or greater than 15.More typically, the number of layers of said droplets is equal to orgreater than 20. In some embodiments, the number of layers is from 10 to50, for instance, from 20 to 30. The number of layers may, for instancebe about 20 or about 28. In other embodiments, the number of layers ofsaid droplets is equal to or greater than 50, for instance, equal to orgreater than 100. The number of layers of said droplets may be equal toor greater than 500, for instance equal to or greater than 1,000. Forinstance, the number of layers of said droplets may be equal to orgreater than 2,000, for instance equal to or greater than 4,000.

The apparatus of the invention may be used to form a first dropletassembly and a second droplet assembly. A third droplet assembly may beproduced, which third droplet assembly comprises the first dropletassembly and the second droplet assembly. The first droplet assembly andthe second droplet assembly may, for instance, be joined together bybilayers formed between at least one droplet of the first dropletassembly and at least one droplet of the second droplet assembly. It isnot necessary for each droplet in the first droplet assembly to form abilayer with each droplet of the second droplet assembly.

In some embodiments, the control unit is adapted to coordinate (a) themovement of the container relative to the or each droplet generator and(b) the dispensing of the droplets, to create a said droplet assemblywhich comprises a plurality of droplets, wherein each of said dropletscomprises (i) an aqueous medium, and (ii) an outer layer of amphipathicmolecules around the surface of the aqueous medium, and wherein each ofsaid droplets contacts another of said droplets to form a bilayer ofsaid amphipathic molecules as an interface between the contactingdroplets, wherein the plurality of droplets comprises a first region ofsaid droplets and a second region of said droplets, wherein each dropletin the first region contacts at least one other droplet in the firstregion to form a bilayer of said amphipathic molecules as an interfacebetween the contacting droplets, and each droplet in the second regioncontacts at least one other droplet in the second region to form abilayer of said amphipathic molecules as an interface between thecontacting droplets, wherein the aqueous medium of the droplets in thefirst region is different from the aqueous medium of the droplets in thesecond region, and/or wherein the composition of the bilayers betweendroplets in the first region is different from the composition of thebilayers between droplets in the second region.

At least one droplet in the first region may, for instance, contact atleast one droplet in the second region to form a bilayer of saidamphipathic molecules as an interface between the contacting droplets.Typically, two or more interfaces between a droplet in the first regionand a droplet in the second region are formed. There may be at least onedroplet in the first region that does not form a bilayer with a dropletin the second region and/or at least one droplet in the second regionthat does not form a bilayer with a droplet in the first region.

In one embodiment, the aqueous medium of the droplets in the firstregion comprises a membrane protein and the aqueous medium of thedroplets in the second region may not comprise said membrane protein;the aqueous media of the droplets in the first and second regions mayhave different concentrations of a membrane protein; the bilayersbetween droplets in the first region may further comprise a membraneprotein, and the bilayers between droplets in the second region eithermay not comprise said membrane protein or may have a lower concentrationof said membrane protein than the bilayers between droplets in the firstregion; the aqueous media of the droplets in the first and secondregions may have different osmolarities; or the aqueous media of thedroplets in the first and second regions may have differentconcentrations of a salt or buffer.

In one embodiment, the aqueous medium of the droplets in the firstregion comprises a first membrane protein and the aqueous medium of thedroplets in the second region comprises a second membrane protein, wherethe first and second membrane proteins are different. The first andsecond membrane proteins may be membrane proteins as defined herein.

Typically, the droplet assembly produced by the apparatus of theinvention comprises at least 100 of said droplets, each of whichcomprises (i) an aqueous medium and (ii) an outer layer of amphipathicmolecules around the surface of the aqueous medium. More typically, thedroplet assembly produced by the apparatus of the invention comprises atleast 1,000 of said droplets, each of which comprises (i) an aqueousmedium and (ii) an outer layer of amphipathic molecules around thesurface of the aqueous medium. Even more typically, the droplet assemblyproduced by the apparatus of the invention comprises at least 10,000 ofsaid droplets (for instance, 25,000), each of which comprises (i) anaqueous medium and (ii) an outer layer of amphipathic molecules aroundthe surface of the aqueous medium.

In some embodiments, the number of droplets in the droplet assemblyproduced by the apparatus of the invention may be very large, forinstance, at least 100,000. The number of droplets in the dropletassembly produced by the apparatus of the invention may be at least1,000,000, for instance, at least 10,000,000. For instance, the numberof droplets in the droplet assembly produced by the apparatus of theinvention may be at least 1,000,000,000. In some embodiments, the numberof droplets in the droplet assembly produced by the apparatus of theinvention may be at least 10,000,000,000, for instance at least50,000,000,000. If, for instance the droplet assembly is a cubecomprising at least 4000 layers, the number of droplets in the dropletassembly produced by the apparatus of the invention may be at least 64billion droplets.

The apparatus of the invention can therefore be adapted to produce athree-dimension droplet assembly with mm-scale geometries. The dropletassembly may, for instance, comprise at least 10,000 of said droplets,wherein each droplet has a diameter of from 10 μm to 1 mm, for instance,from 10 μm to 200 μm. Typically, each droplet has a diameter of from 30μm to 60 μm, for instance, each droplet has a diameter of about 50 μm.The droplet assembly may be functionalised, for instance, through theuse of membrane proteins in a selected region(s) of the assembly.Additionally or alternatively, the droplet assembly can be made to foldusing osmosis.

The apparatus of the invention may be adapted to produce athree-dimension droplet assembly with cm-scale geometries, or larger.

Three-dimensional droplet assemblies may be gelled together to form anew droplet assembly. In this way, individual droplet assemblies may beused as building bricks, for instance, to form complex structures.

In some embodiments, the control unit is adapted to coordinate (a) themovement of the container relative to the or each droplet generator and(b) the dispensing of the droplets, to produce a droplet assembly by aprocess as defined below for the process of the invention.

In other embodiments, the control unit is adapted to coordinate (a) themovement of the container relative to the or each droplet generator and(b) the dispensing of the droplets, to produce a droplet assembly asdefined below for the product aspect of the invention.

In some embodiments of the apparatus of the invention, when thecontainer contains a hydrophobic medium, said hydrophobic medium is adrop of a hydrophobic medium within a second bulk medium which is anaqueous medium, and said container contains said second bulk mediumwhich is an aqueous medium and the drop of the hydrophobic medium. Thesecond bulk medium which is an aqueous medium may be the same ordifferent from the aqueous media defined previously.

Usually, the drop of the hydrophobic medium further comprises aperipheral layer of amphipathic molecules around the surface of thedrop, as an interface between the drop and the second bulk medium whichis an aqueous medium. The amphipathic molecules may be the sameamphipathic molecules as the amphipathic molecules in the outer layer ofamphipathic molecules around the surface of the aqueous medium or theymay be different amphipathic molecules. Typically, a bilayer ofamphipathic molecules is formed at the surface of the drop. There maytherefore be (i) droplet interface bilayers between droplets and (ii)bilayers between droplets and the surface of the drop. These bilayersallow communication (i) between droplets within the droplet assembly and(ii) between a droplet within the droplet assembly and the externalenvironment.

The apparatus of the invention may be used in the process of theinvention.

The invention also relates to a process for producing a droplet assemblyusing an apparatus for producing the droplet assembly, which dropletassembly comprises: a plurality of droplets, wherein each of saiddroplets comprises: (i) a droplet medium, and (ii) an outer layer ofamphipathic molecules around the surface of the droplet medium, whereinthe droplet medium is an aqueous medium or a hydrophobic medium, andwherein at least one of said droplets contacts another of said dropletsto form a layer of said amphipathic molecules as an interface betweenthe contacting droplets; which apparatus comprises: at least one dropletgenerator; a container which is moveable relative to the at least onedroplet generator; and a control unit, which control unit is adapted tocontrol the dispensing of droplets from the at least one dropletgenerator and the movement of the container relative to the at least onedroplet generator; wherein said container of the apparatus contains abulk medium, wherein: when the droplet medium is an aqueous medium thebulk medium is a hydrophobic medium, and when the droplet medium is ahydrophobic medium the bulk medium is an aqueous medium; which processcomprises: (a) a plurality of dispensing steps, wherein each dispensingstep comprises dispensing a droplet of the droplet medium from a saiddroplet generator into the bulk medium, in the presence of amphipathicmolecules, and thereby forming in the bulk medium a droplet whichcomprises (i) said droplet medium and (ii) an outer layer of amphipathicmolecules around the surface of the droplet medium; and (b) moving thecontainer relative to the at least one droplet generator, to control therelative positioning of the droplets in the bulk medium.

The layer of said amphipathic molecules which is an interface betweencontacting droplets may, for instance, be a bilayer of said amphipathicmolecules or a layer of a block copolymer. The layer of a blockcopolymer may, for example, be a layer of a triblock copolymer. When thedroplet medium is an aqueous medium the layer of said amphipathicmolecules which is an interface between contacting droplets may, forinstance, be a bilayer of said amphipathic molecules or a layer of ablock copolymer. When the droplet medium is a hydrophobic medium thelayer of said amphipathic molecules which is an interface betweencontacting droplets may, for instance, be a bilayer of said amphipathicmolecules or a layer of a block copolymer.

Typically, the layer of said amphipathic molecules which is an interfacebetween contacting droplets is a bilayer of said amphipathic molecules.

The droplet medium is typically an aqueous medium and the bulk medium istypically a hydrophobic medium. Another embodiment is envisaged howeverin which the droplet medium is a hydrophobic medium and the bulk mediumis an aqueous medium.

Usually, however, the droplet medium is an aqueous medium and the bulkmedium is a hydrophobic medium, and the invention will generally bedescribed hereinbelow in these terms. However, as the skilled personwill appreciate, any of the embodiments of the invention describedherein in those terms may also be performed “in reverse”, using ahydrophobic medium as the droplet medium instead of an aqueous medium,and using an aqueous medium as the bulk medium instead of a hydrophobicmedium.

The aqueous medium is typically an aqueous medium as further definedhereinbefore for the apparatus of the invention.

Typically, each of the plurality of droplets has a diameter of equal toor less than 1 mm, for instance, from 10 μm to 1 mm. Usually, each ofthe plurality of droplets has a diameter of from 10 μm to 200 μm, forinstance, of from 30 μm to 60 μm. More typically, each of the pluralityof droplets has a diameter of about 50 μm.

The amphipathic molecules may be any suitable amphipathic molecules.They may, for instance, be amphipathic molecules as defined hereinabovefor the apparatus of the invention.

The amphipathic molecules may, for instance, be disposed in the aqueousmedium or in the hydrophobic medium. Typically, the amphipathicmolecules are disposed in the hydrophobic medium.

When the aqueous medium is dispensed into the hydrophobic medium in thepresence of amphipathic molecules, an aqueous droplet forms, whichdroplet comprises (i) an aqueous medium and (ii) an outer layer ofamphipathic molecules around the surface of the aqueous medium.

A droplet may, in some embodiments, comprise other materials, compoundsor substances. For instance, a droplet may comprise a small molecule,such as a dye, or a magnet. Suitable dyes include, but are not limitedto, xylene cyanol FF, orange G, pyranine, fluorescein and 5-cTAMRA(5-carboxytetramethylrhodamine). Alternatively, a droplet may compriseany suitable sensor molecule, for instance a sensor molecule that issensitive to a particular chemical or is a light-sensitive molecule, orit may be sensitive to changes in pH or temperature. In someembodiments, the sensor molecule may be sensitive to the presence (orabsence) of a particular biochemical or enzyme. As a furtheralternative, a droplet may comprise a therapeutic agent, such as aprodrug, or a diagnostic agent, such as a contrast agent. A droplet maycomprise an enzyme.

A droplet may comprise a living cell (or living cells), for example foruse in tissue engineering. For instance, the cells may be allowed togrow inside the droplet after printing and/or to break down the bilayersbetween droplets after printing.

A droplet assembly, once printed, may be used as a template for thepatterning of a solid material. The solid material may, for instance, beused in electronics, optics, photonics, or other material scienceapplications. A droplet, or droplets, may, for instance, compriseinorganic materials that could diffuse between specific droplets. Theinorganic materials may then react to form inorganic solids such ascadmium sulphide.

The process may, for instance, comprise moving the container, to controlthe relative positioning of the droplets in the hydrophobic mediumand/or moving the at least one droplet generator, to control therelative positioning of the droplets in the hydrophobic medium.

Usually, the container is a container as defined hereinabove for theapparatus of the invention.

The or each droplet generator may, for instance, be a droplet generatoras defined hereinabove for the apparatus of the invention.

Typically, the control unit is a control unit as defined hereinabove forthe apparatus of the invention. Usually, the control unit controls thedispensing steps, and the movement of the container relative to the atleast one droplet generator.

Usually, the control unit coordinates the dispensing steps, and themovement of the container relative to the at least one dropletgenerator, to produce the droplet assembly.

Typically, moving the container relative to the at least one dropletgenerator comprises: moving the container relative to the at least onedroplet generator to position at least one of said droplets adjacent toanother of said droplets so that at least one of said droplets contactsanother of said droplets to form a layer of said amphipathic moleculesas an interface between the contacting droplets.

The layer of said amphipathic molecules which is an interface betweencontacting droplets may, for instance, be a bilayer of said amphipathicmolecules or a layer of a block copolymer. The layer of a blockcopolymer may, for example, be a layer of a triblock copolymer. When thedroplet medium is an aqueous medium the layer of said amphipathicmolecules which is an interface between contacting droplets may, forinstance, be a bilayer of said amphipathic molecules or a layer of ablock copolymer. When the droplet medium is a hydrophobic medium thelayer of said amphipathic molecules which is an interface betweencontacting droplets may, for instance, be a bilayer of said amphipathicmolecules or a layer of a block copolymer.

Typically, the layer of said amphipathic molecules which is an interfacebetween contacting droplets is a bilayer of said amphipathic molecules.

Moving the container relative to the at least one droplet generatortypically comprises: moving the container relative to the at least onedroplet generator to position each droplet adjacent to at least oneother droplet.

Usually, the droplet assembly comprises said plurality of droplets andeach of said droplets contacts another of said droplets to form abilayer of said amphipathic molecules as an interface between thecontacting droplets.

Moving the container relative to the at least one droplet generator may,for instance, comprise: moving the container relative to the at leastone droplet generator to position each droplet adjacent to at least oneother droplet, so that each of said droplets contacts another of saiddroplets to form a layer of said amphipathic molecules as an interfacebetween contacting droplets.

The layer of said amphipathic molecules which is an interface betweencontacting droplets may, for instance, be a bilayer of said amphipathicmolecules or a layer of a block copolymer. The layer of a blockcopolymer may, for example, be a layer of a triblock copolymer. When thedroplet medium is an aqueous medium the layer of said amphipathicmolecules which is an interface between contacting droplets may, forinstance, be a bilayer of said amphipathic molecules or a layer of ablock copolymer. When the droplet medium is a hydrophobic medium thelayer of said amphipathic molecules which is an interface betweencontacting droplets may, for instance, be a bilayer of said amphipathicmolecules or a layer of a block copolymer.

Typically, the layer of said amphipathic molecules which is an interfacebetween contacting droplets is a bilayer of said amphipathic molecules.

Usually, moving the container relative to the at least one dropletgenerator comprises: moving the container relative to the at least onedroplet generator between dispensing steps.

For instance, the process usually comprises (b) moving the containerrelative to the at least one droplet generator between at least two ofthe dispensing steps. In some embodiments, the process comprises (b)moving the container relative to the at least one droplet generatorbetween at least 50% of the dispensing steps, for instance between atleast 80%. The process may, for instance, comprises (b) moving thecontainer relative to the at least one droplet generator betweensubstantially all of the dispensing steps. For instance, the process maycomprise comprises (b) moving the container relative to the at least onedroplet generator between all of the dispensing steps.

Although it may necessary to move the container relative to the at leastone droplet generator between dispensing steps, it is not alwaysnecessary to move the container relative to the at least one dropletgenerator between dispensing steps. For instance, when the same dropletgenerator dispenses two droplets, one directly after the other, whereone droplet is to be dispensed directly on top of the other droplet, thecontainer does not need to be moved relative to the droplet generator inorder that the droplets be dispensed at the correct location. Similarly,two droplets may be dispensed one after the other from two differentdroplet generators and the positioning of the two droplet generators maybe such that the container does not need to be moved relative to the twodroplet generators in order that the droplets be dispensed at thecorrect location.

In some embodiments, moving the container relative to the at least onedroplet generator comprises: moving the container relative to the atleast one droplet generator between dispensing steps and duringdispensing steps.

For instance, the process may comprise (b) moving the container relativeto the at least one droplet generator between and during at least two ofthe dispensing steps. The process may, for instance, comprise (b) movingthe container relative to the at least one droplet generator between andduring at least 50% of the dispensing steps, for instance between andduring at least 80%. In some embodiments, the process comprises (b)moving the container relative to the at least one droplet generatorbetween and during substantially all of the dispensing steps, forinstance between and during all of the dispensing steps.

Typically, the droplet medium is an aqueous medium and the bulk mediumis a hydrophobic medium. Thus, usually, the process of the invention isa process for producing a droplet assembly using an apparatus forproducing the droplet assembly, which droplet assembly comprises: aplurality of droplets, wherein each of said droplets comprises: (i) anaqueous medium, and (ii) an outer layer of amphipathic molecules aroundthe surface of the aqueous medium, and wherein at least one of saiddroplets contacts another of said droplets to form a bilayer of saidamphipathic molecules as an interface between the contacting droplets;which apparatus comprises: at least one droplet generator; a containerwhich is moveable relative to the at least one droplet generator; and acontrol unit, which control unit is adapted to control the dispensing ofdroplets from the at least one droplet generator and the movement of thecontainer relative to the at least one droplet generator; wherein saidcontainer of the apparatus contains a hydrophobic medium; which processcomprises: (a) a plurality of dispensing steps, wherein each dispensingstep comprises dispensing a droplet of an aqueous medium from a saiddroplet generator into the hydrophobic medium, in the presence ofamphipathic molecules, and thereby forming in the hydrophobic medium adroplet which comprises (i) said aqueous medium and (ii) an outer layerof amphipathic molecules around the surface of the aqueous medium; and(b) moving the container relative to the at least one droplet generator,to control the relative positioning of the droplets in the hydrophobicmedium.

Typically, the process of the invention comprises: (a) a firstdispensing step, comprising dispensing a droplet of an aqueous mediumfrom a said droplet generator into the hydrophobic medium, in thepresence of amphipathic molecules, and thereby forming in thehydrophobic medium a first droplet, which first droplet comprises (i)said aqueous medium and (ii) an outer layer of amphipathic moleculesaround the surface of the aqueous medium; (b) moving the containerrelative to the at least one droplet generator, to control thepositioning of a second droplet in the hydrophobic medium relative tothe first droplet in the hydrophobic medium; and (c) a second dispensingstep, comprising dispensing a droplet of an aqueous medium from a saiddroplet generator into the hydrophobic medium, in the presence ofamphipathic molecules, and thereby forming in the hydrophobic medium asecond droplet, which second droplet comprises (i) said aqueous mediumand (ii) an outer layer of amphipathic molecules around the surface ofthe aqueous medium.

The droplet generator of the first dispensing step may be the samedroplet generator as the droplet generator of the second dispensing stepor it may be a different droplet generator.

As mentioned above, for the apparatus of the invention, the size of thedroplet may be controlled by, for example, the droplet generator fromwhich it is dispensed. When the droplet generator of the firstdispensing step is different from the droplet generator of the seconddispensing step, the first droplet may have the same diameter as thesecond droplet or it may have a different diameter.

Typically, the diameter of the first droplet will be equal to or lessthan 1 mm, for instance, from 10 μm to 1 mm. Usually, the diameter ofthe first droplet will be from 10 μm to 200 μm, for instance, of from 30μm to 60 μm. More typically, the diameter of the first droplet will beabout 50 μm.

Usually, the diameter of the second droplet will be equal to or lessthan 1 mm, for instance, from 10 μm to 1 mm. Typically, the diameter ofthe second droplet will be from 10 μm to 200 μm, for instance, of from30 μm to 60 μm. In some embodiments, the diameter of the second dropletis about 50 μm.

The aqueous medium of the first droplet may be the same as the aqueousmedium of the second droplet or it may be different. When the dropletgenerator of the first dispensing step is the same as the dropletgenerator of the second dispensing step, the aqueous medium of the firstdroplet is typically the same as the aqueous medium of the seconddroplet. When the droplet generator of the first dispensing step isdifferent from the droplet generator of the second dispensing step, theaqueous medium of the first droplet may, for instance, be different tothe aqueous medium of the second droplet.

The amphipathic molecules of the first droplet may be the same as theamphipathic molecules of the second droplet or they may be different.Typically, the amphipathic molecules are amphipathic molecules asdefined herein for the apparatus of the invention.

In some embodiments, said (b) moving the container relative to the atleast one droplet generator comprises: moving the container relative tothe at least one droplet generator so that the second droplet ispositioned adjacent to the first droplet, so that the first and seconddroplets contact one another to form a bilayer of said amphipathicmolecules as an interface between the contacting droplets.

Typically, the process further comprises: (d) moving the containerrelative to the at least one droplet generator, to control thepositioning of a further droplet in the hydrophobic medium relative tothe other droplets in the hydrophobic medium; and (e) a furtherdispensing step, comprising dispensing a droplet of an aqueous mediumfrom a said droplet generator into the hydrophobic medium, in thepresence of amphipathic molecules, and thereby forming in thehydrophobic medium a further droplet, which further droplet comprises(i) said aqueous medium and (ii) an outer layer of amphipathic moleculesaround the surface of the aqueous medium.

The aqueous medium of the further droplet is usually an aqueous mediumas further defined herein.

Typically, the amphipathic molecules are amphipathic molecules asdefined herein for the apparatus of the invention.

Usually, said (d) moving the container relative to the at least onedroplet generator, comprises: moving the container relative to the atleast one droplet generator so that the further droplet is positionedadjacent to at least one other droplet in the hydrophobic medium, sothat the further droplet contacts at least one other droplet in thehydrophobic medium to form a bilayer of said amphipathic molecules as aninterface between the contacting droplets.

In some embodiments, the process of the invention comprises at least 500of said further dispensing steps (e). Typically, the process comprises1,000 of said further dispensing steps (e), for instance, 5,000 of saidfurther dispensing steps (e). For instance, the process may comprise atleast 10,000 of said further dispensing steps (e). In some embodiments,the process comprises at least 25,000 of said further dispensing steps(e).

Usually, the process comprises at least 500 of said steps of (d) movingthe container relative to the at least one droplet generator, and atleast 500 of said further dispensing steps (e). For instance, theprocess may comprise at least 1,000 of said steps of (d) moving thecontainer relative to the at least one droplet generator, and at least1,000 of said further dispensing steps (e). In some embodiments, theprocess may, for instance, comprise at least 5,000 of said steps of (d)moving the container relative to the at least one droplet generator, andat least 5,000 of said further dispensing steps (e).

Typically, the process comprises at least 10,000 of said steps of (d)moving the container relative to the at least one droplet generator, andat least 10,000 of said further dispensing steps (e). In someembodiments, the process comprises at least 25,000 of said steps of (d)moving the container relative to the at least one droplet generator, andat least 25,000 of said further dispensing steps (e).

The process may comprise at least 1,000,000 of said steps of (d) movingthe container relative to the at least one droplet generator, and atleast 1,000,000 of said further dispensing steps (e). For instance, theprocess may comprise at least 1,000,000,000 of said steps of (d) movingthe container relative to the at least one droplet generator, and atleast 1,000,000,000 of said further dispensing steps (e).

Usually, the plurality of dispensing steps comprises dispensing stepswhich together produce a row of said droplets in the hydrophobic medium,wherein each droplet in the row contacts another droplet in the row toform a bilayer of said amphipathic molecules as an interface betweencontacting droplets.

Typically, the number of droplets in the row is at least 5, forinstance, at least 10. More typically, the number of droplets in the rowis at least 20, for instance, at least 50. Usually, the number ofdroplets in the row is from 5 to 500, for instance, from 10 to 250. Insome embodiments, the number of droplets in the row is from 20 to 100.For instance, the number of droplets in the row may be from 20 to 35.

In some embodiments, the number of droplets in the row is at least 500,for instance, at least 1,000. The number of droplets in the row may, forinstance, be at least 2,000, for instance, at least 4,000. The number ofdroplets in the row may, for instance, be from 5 to 5,000.

A plurality of rows of droplets may be disposed adjacent to one anotherto form a layer. In a layer, the plurality of rows are typically incontact with one another, so that droplets in one row contact dropletsin an adjacent row or rows to form bilayers of said amphipathicmolecules as interfaces between contacting droplets in adjacent rows. Alayer may for instance comprise at least 10 such rows disposed adjacentto one another, or for instance at least 20 such rows disposed adjacentto one another. In some embodiments, a layer comprises at least 50 suchrows disposed adjacent to one another, or for instance at least 100 suchrows disposed adjacent to one another. The number of droplets in the rowmay be as defined above. A rectangular layer may for instance be a layerof at least 20 droplets by at least 20 droplets (i.e. a layer of atleast 20×20 droplets). However, each row need not be the same length,and so a variety of different layer shapes can be formed by disposingrows of different lengths adjacent to one another. Self-folding dropletassemblies, for instance, may comprise rows of different lengths, asdiscussed in the Examples section.

In some embodiments, the plurality of dispensing steps comprises: afirst set of dispensing steps which together produce a first row of saiddroplets in the hydrophobic medium, wherein each droplet in the firstrow contacts another droplet in the first row to form a bilayer of saidamphipathic molecules as an interface between contacting droplets; and asecond set of dispensing steps which together produce a second row ofsaid droplets in the hydrophobic medium, wherein each droplet in thesecond row contacts another droplet in the second row to form a bilayerof said amphipathic molecules as an interface between contactingdroplets.

Typically, droplets in the second row are disposed adjacent to dropletsin the first row, so that droplets in the first row contact droplets inthe second row to form bilayers of said amphipathic molecules asinterfaces between the contacting droplets.

Usually, the plurality of dispensing steps comprises dispensing stepswhich together produce a plurality of rows of said droplets in thehydrophobic medium, wherein each droplet in a row contacts at least oneother droplet in the row to form a bilayer of said amphipathic moleculesas an interface between contacting droplets, and wherein each row isdisposed adjacent to another row, so that droplets in a row contactdroplets in an adjacent row to form bilayers of amphipathic molecules asinterfaces between the contacting droplets.

The number of rows disposed adjacent to one another may be as definedabove.

In some embodiments, the plurality of dispensing steps comprisesdispensing steps which together produce a layer of said droplets in thehydrophobic medium, wherein each droplet in the layer contacts at leastone other droplet in the layer to form a bilayer of said amphipathicmolecules as an interface between contacting droplets.

A layer generally comprises a plurality of rows of droplets disposedadjacent to one another, and in contact with one another, so thatdroplets in a row contact droplets in an adjacent row or rows to formbilayers of said amphipathic molecules as interfaces between contactingdroplets. A layer may for instance comprise at least 20 such rowsdisposed adjacent to one another. The number of droplets in the row maybe as defined above. Different rows in the layer may have differentnumbers of droplets and so the layer need not be rectangular, but may beany shape. For instance, a layer can be rectangular, circular, oval,diamond shaped, or any other two-dimensional shape. Also, a layer mayhave one or more gaps (i.e. places where no droplet is present) at acertain position or positions. Thus, a layer may for instance bering-shaped, or frame-shaped. A layer which is rectangular may forinstance be a layer of at least 20 droplets by at least 20 droplets(i.e. a 20×20 droplet layer). Typically, one layer is above or belowanother layer. Usually, the droplet assembly is built upwards inhorizontal layers.

By disposing layers of droplets on top of one another, dropletassemblies having a wide variety of three-dimensional (3D) shapes can beconstructed. One of the simplest 3D droplet assemblies has a cuboidshape. However, by disposing layers of different shapes on top of oneanother, a wide variety of other three-dimensional droplet assemblystructures can be formed, such as, for instance, a droplet assemblyhaving a pyramidal shape or for instance having the shape of a prism.

Typically, the plurality of dispensing steps comprises: a first set ofdispensing steps which together produce a first layer of said dropletsin the hydrophobic medium, wherein each droplet in the first layercontacts at least one other droplet in the first layer to form a bilayerof said amphipathic molecules as an interface between contactingdroplets; and a second set of dispensing steps which together produce asecond layer of said droplets in the hydrophobic medium, wherein eachdroplet in the second layer contacts at least one other droplet in thesecond layer to form a bilayer of said amphipathic molecules as aninterface between contacting droplets.

Usually, droplets in the second layer are disposed adjacent to dropletsin the first layer, so that droplets in the first layer contact dropletsin the second layer to form bilayers of said amphipathic molecules asinterfaces between the contacting droplets.

More typically, droplets in the second layer are disposed on droplets inthe first layer, so that droplets in the first layer contact droplets inthe second layer to form bilayers of said amphipathic molecules asinterfaces between the contacting droplets.

Generally, the plurality of dispensing steps comprises dispensing stepswhich together produce a plurality of layers of said droplets in thehydrophobic medium, wherein each droplet in a layer contacts at leastone other droplet in the layer to form a bilayer of said amphipathicmolecules as an interface between contacting droplets, and wherein eachlayer is disposed adjacent to another layer, so that droplets in a layercontact droplets in an adjacent layer to form bilayers of amphipathicmolecules as interfaces between the contacting droplets.

Usually, each layer is disposed adjacent to another layer, so thatdroplets in a layer contact droplets in another layer to form bilayersof amphipathic molecules as interfaces between the contacting droplets.

More typically, each layer is disposed on another layer, so thatdroplets in a layer contact droplets in another layer to form bilayersof amphipathic molecules as interfaces between the contacting droplets.

In some embodiments, droplets in a first layer contact droplets insecond layer to form bilayers of amphipathic molecules as interfacesbetween the contacting droplets, but droplets within the second layerare not in contact with each other. This may occur if, for instance, thedroplet in the second layer have a smaller diameter than droplet in thefirst layer. It may also occur if, for instance, gaps (i.e. spaceswithout droplets) are specifically left in the second layer.

The layer may, for instance, be on one layer and below another layer.

The number of layers of said droplets may, for instance, be equal to orgreater than 5. Typically, the number of layers of said droplets isequal to or greater than 10, for instance, equal to or greater than 15.More typically, the number of layers of said droplets is equal to orgreater than 20, for instance, equal to or greater than 25. In someembodiments, the number of layers of said droplets is equal to orgreater than 50, for instance, equal to or greater than 100. In otherembodiments, the number of layers is from 10 to 50, for instance, from20 to 30.

In some embodiments, the number of layers is at least 500, for instance,at least 1,000. The number of layers may, for instance, be at least2,000, for instance, at least 4,000. The number of layers may, forinstance, be from 5 to 5,000.

The process of the invention therefore provides an effective process forproducing droplet assemblies comprising a large number of droplets, thathave, for instance, mm-scale geometries.

The process of the invention may be used to produce a three-dimensiondroplet assembly with cm-scale geometries, or larger.

As discussed in the Examples, under Supplementary Methods, the printingpattern used to produce the droplet assembly can be controlled tocontrol the accuracy of the assemblies that are printed. For instance,when the container is moved relative to the at least one dropletgenerator, the motion can cause displacement of droplets that have beenrecently dispensed. When, for instance, the tip of the droplet generatoris immersed in a hydrophobic medium, the movement of the tip relativethe hydrophobic medium creates a drag force. It may, for example, beadvantageous to allow recently-dispensed droplets enough time to comeinto contact with another droplet before the container is moved relativeto the at least one droplet generator. However, if too many pauses arebuilt into the process, a large assembly comprising thousands ofdroplets may take a very long time to produce. The inventors have foundthat, in some embodiments, a pause after the printing of each row canallow a significant reduction in printing time without a significantcost to print quality (see Supplementary Discussion).

There are several ways in which the overall printing rate may becontrolled. The overall printing rate may, for example, be controlled bydispensing droplets from two or more droplet generators simultaneously.

An individual droplet may be displaced by movement of the dropletgenerator relative to the container. It may therefore be desirable toallow each droplet, or at least each row of droplets, to reach itsintended position in the network before the droplet generator is movedrelative to the container. The printing rate could be changed by makingdroplets sink more quickly, which could be achieved by: (i) decreasingthe viscosity of the hydrophobic medium; (ii) decreasing the density ofthe hydrophobic medium; (iii) increasing the density of the aqueousmedium; or (iv) increasing the diameter of the droplets dispensed fromthe or each droplet generator. For example, lowering the viscosity ofthe hydrophobic medium would decrease the displacement of the dropletcaused by the relative motion of the droplet generator to the containerand increase the sink rate. Typically, this will mean the dropletgenerator can be moved to the next position more quickly.

Alternatively, the printing rate may be changed by increasing ordecreasing the distance between the tip of the or each droplet generatorand the position at which the droplet is to come to rest.

In one embodiment, each droplet is dispensed at least 100 μm, forinstance, at least 150 μm, above the position at which it is to come torest. Typically, each droplet is dispensed at least 200 μm above theposition at which it is to come to rest. The inventors have found thatwhen droplet production is attempted at smaller distances that these,droplet formation may be hindered. However, the smaller distances may besuitable if, for example, the viscosity or density of the hydrophobicmedium is changed, or the density of the aqueous medium is changed.

The outer layer of amphipathic molecules to needs form before thedispensed droplet comes into contact with another droplet. This can becontrolled by, for example: (a) increasing the concentration ofamphipathic molecules in the hydrophobic medium or the aqueous medium;(b) decreasing the diameter of the droplet; or (c) using a hydrophobicmedium that encourages rapid monolayer formation. The inventors havefound that rapid monolayer formation may be encouraged when thehydrophobic medium comprises a silicon oil and/or a hydrocarbon such ashexadecane. The monolayer may, for instance, form within about 1 s ofdroplet formation.

Typically, the process of the invention further comprises a delay,between a dispensing step and a step of moving the container relative tothe at least one droplet generator, for reducing droplet slippage.

Usually, the delay is equal to or greater than 1 ms, for instance, equalto or greater than 10 ms. Typically, the delay is equal to or greaterthan 25 ms, for instance, equal to or greater than 50 ms. For instance,the delay may be about 50 ms.

In some embodiments, the delay is after a row has been produced. Theprocess may, for example, comprise: a first set of dispensing stepswhich together produce a first row of said droplets in the hydrophobicmedium, wherein each droplet in the first row contacts another dropletin the first row to form a bilayer of said amphipathic molecules as aninterface between contacting droplets; a second set of dispensing stepswhich together produce a second row of said droplets in the hydrophobicmedium, wherein each droplet in the second row contacts another dropletin the second row to form a bilayer of said amphipathic molecules as aninterface between contacting droplets; and a delay between the first setof dispensing steps and the second set of dispensing steps. Usually, thedelay is equal to or greater than 1 second. For instance, the delay maybe about 2 seconds.

In some embodiments, the plurality of dispensing steps may comprise atleast one set of dispensing steps which together produce a row of saiddroplets in the hydrophobic medium, wherein each droplet in the rowcontacts another droplet in the row to form a bilayer of saidamphipathic molecules as an interface between contacting droplets, andwherein the process further comprises a said delay between (i) the lastdispensing step in the set of dispensing steps which together producethe row of said droplets, and (ii) a subsequent step of moving thecontainer relative to the at least one droplet generator.

Usually, the process further comprises said delay between the lastdispensing step in each set of dispensing steps which together produce arow of said droplets, and a subsequent step of moving the containerrelative to the at least one droplet generator.

Typically, the hydrophobic medium, or the aqueous medium, or both,further comprise the amphipathic molecules. The amphipathic moleculesare usually as defined herein for the apparatus of the invention.

In some embodiments, one or more additional droplets are dispensed atthe end of a row. The one or more additional droplets may, for instance,prevent internal droplets in the droplet assembly from rolling out oftheir intended boundary.

Usually, the at least one droplet generator is a piezoelectric dropletgenerator which comprises a piezoelectric transducer for dispensingdroplets, and wherein each dispensing step comprises the application ofa voltage pulse to the piezoelectric component. The control unittypically controls the application of the voltage pulses to thepiezoelectric component.

Typically, the voltage pulse has a peak-to-peak amplitude of from 5 V to100 V, for instance, of from 20 V to 60 V.

Usually, each pulse has a duration of from 10 to 1,500 μs, for instance,of from 100 to 800 μs.

Typically, the voltage pulse is a square voltage pulse.

As shown in FIG. 10, the diameter of the droplet may be tuned by varyingthe amplitude and duration of the voltage pulses.

The process of the invention may further comprise applying small voltagepulses (e.g. from 6V to 18V voltage pulses, more typically approximately12 V voltage pulses) periodically, e.g. approximately every ˜10-30 s.Such pulses can advantageously ensure that the droplet medium (e.g.aqueous medium) does not gradually bud out of the tip of the printingnozzle and leak into the oil (since the aqueous chamber is typicallyplaced higher than the oil container and can therefore exert greaterhydrostatic pressure than the oil at the nozzle tip). It has been foundthat applying these low-voltage pulses can return the aqueous-oilinterface at the nozzle tip to a planar geometry, without causing adroplet to be ejected.

Usually, the droplets are dispensed at a rate of from 0.01 to 100 s⁻¹,for instance, at a rate of from 0.01 to 50 s⁻¹. Typically, the dropletsare dispensed at a rate of from 0.01 to 10 s⁻¹, for instance, at a rateof from 0.5 to 5 s⁻¹. In some embodiments, the droplets are dispensed ata rate of about 1 s⁻¹.

Typically, in the process of the invention, the apparatus comprises twoor more of said droplet generators. As mentioned above, differentdroplet generators may dispense droplets having different diameters ordroplets comprising different aqueous media.

In some embodiments, the droplet medium is an aqueous medium and thebulk medium is a hydrophobic medium, and the plurality of dispensingsteps comprises: a first set of dispensing steps, which together producea first region of said droplets in the hydrophobic medium, wherein eachdroplet in the first region contacts at least one other droplet in thefirst region to form a bilayer of said amphipathic molecules as aninterface between contacting droplets; and a second set of dispensingsteps, which together produce a second region of said droplets in thehydrophobic medium, wherein each droplet in the second region contactsat least one other droplet in the second region to form a bilayer ofsaid amphipathic molecules as an interface between contacting droplets.

As the skilled person will appreciate, entire regions are not usuallyprinted sequentially. Indeed, for a given layer, the droplets of thefirst region for that particular layer are often printed together,followed by all the droplets of the second region in that particularlayer. Printing of another layer may then commence, which may involveprinting of further droplets for the first region and further dropletsfor the second region (not necessarily in that order). Thus, thedispensing steps of the first set do not necessarily all take placesequentially, and likewise the dispensing steps of the second set do notnecessarily all take place sequentially. Also, the dispensing steps ofthe second set do not necessarily all take place after the dispensingsteps in the first set, and vice versa.

The first and second regions can be virtually any two- orthree-dimensional structure that can be made from droplets using theprocess of the invention.

Usually, the first region and/or the second region comprises at least100 droplets, for instance at least 500 droplets. In some embodiments,the first region and/or the second region comprises at least 1,000droplets, for instance at least 5,000 droplets.

The number of droplets in each of the first and second regions may bevery large, for instance, at least 100,000. The number of droplets ineach of the first and second regions may be at least 1,000,000, forinstance, at least 10,000,000. For instance, the number of droplets ineach of the first and second regions may be at least 1,000,000,000. Insome embodiments, the number of droplets in each of the first and secondregions may be at least 10,000,000,000, for instance at least50,000,000,000.

Typically, in the process of the invention, the apparatus comprises afirst droplet generator and a second droplet generator, wherein thedroplets dispensed in said first set of dispensing steps are dispensedfrom the first droplet generator into the hydrophobic medium, and thedroplets dispensed in said second set of dispensing steps are dispensedfrom the second droplet generator into the hydrophobic medium. Asmentioned above, different droplet generators may dispense dropletshaving different diameters and/or droplets comprising different aqueousmedia.

Also, as mentioned above, entire regions are not always printedsequentially. Thus, for a given layer, the droplets of the first regionfor that particular layer are often printed together, followed by allthe droplets of the second region in that particular layer. Printing ofanother layer may then commence, which may involve printing of furtherdroplets for the first region and further droplets for the second region(not necessarily in that order). Thus, in the embodiment of theinvention defined above, the dispensing steps of the first set do notnecessarily all take place sequentially, and likewise the dispensingsteps of the second set do not necessarily all take place sequentially.Also, the dispensing steps of the second set do not necessarily all takeplace after the dispensing steps in the first set, and vice versa.

Usually, droplets in the second region are disposed adjacent to dropletsin the first region, so that droplets in the first region contactdroplets in the second region to form bilayers of said amphipathicmolecules as interfaces between the contacting droplets.

Typically some, but not all, of the droplets in the second region aredisposed adjacent to droplets in the first region. Thus, typically onlysome of the droplets in the first region contact droplets in the secondregion to form bilayers. This will often be the case if the first and/orsecond region is a three-dimensional structure made up of a plurality oflayers of droplets. In such cases, only droplets on the surface of thefirst region will be able to contact droplets in the second region.

Usually, the first region of droplets comprises a row of droplets, partof a row of droplets, a plurality of rows of droplets, part of aplurality of rows of droplets, a layer of droplets, part of a layer ofdroplets, a plurality of layers of droplets, or part of a plurality oflayers of droplets; and the second region of droplets comprises a row ofdroplets, part of a row of droplets, a plurality of rows of droplets,part of a plurality of rows of droplets, a layer of droplets, part of alayer of droplets, a plurality of layers of droplets, or part of aplurality of layers of droplets.

The first and second regions may be first and second rows as definedhereinabove. Additionally or alternatively, the first and second regionsmay be first and second layers as defined hereinabove.

In some embodiments, the first region of droplets forms a pathway ofdroplets through the second region of droplets. This is illustrated inFIGS. 12A, 12B, 12D.

In some embodiments, the aqueous medium of the droplets dispensed intothe hydrophobic medium in said first set of dispensing steps furthercomprises a membrane protein, and wherein the aqueous medium of thedroplets dispensed into the hydrophobic medium in said second set ofdispensing steps does not comprise said membrane protein.

The membrane protein may be of any type. The membrane protein may forinstance be a membrane pump, channel and/or pore, to allow for precisecontrol over the exchange of material, and electrical communication,between (i) individual droplets within the assembly and (ii) the dropletassembly and an external solution. The membrane protein could forinstance be an αHL pore. However, any suitable membrane protein can beused including one from the two major classes, that is, β-barrels orα-helical bundles. Besides a protein pore or channel, further possiblemembrane proteins include, but not exclusively, a receptor, atransporter or a protein which effects cell recognition or acell-to-cell interaction. The channel can be a voltage-gated ionchannel, a light-sensitive channel such as bacteriorhodopsin, aligand-gated channel or a mechano-sensitive channel. Typically, themembrane protein is an α-hemolysin (αHL) pore.

Other suitable membrane protein include, but are not limited to,bacterial peptides and ionophores. The membrane protein may,alternatively, be an engineered membrane protein or synthetic membraneprotein. The engineered membrane protein may, for instance, be agenetically engineered protein, or a covalent or non-covalent chemicallyengineering protein. The synthetic membrane protein may, for instance,be a peptide or an organic molecule.

Usually, the bilayers of amphipathic molecules formed between thecontacting droplets in the first region further comprise said membraneprotein.

In some embodiments, the bilayers of amphipathic molecules formedbetween contacting droplets in the second region do not comprise saidmembrane protein.

In other embodiments, the aqueous medium of the droplets dispensed intothe hydrophobic medium in said first set of dispensing steps comprises ahigher concentration of a membrane protein than the aqueous medium ofthe droplets dispensed into the hydrophobic medium in said second set ofdispensing steps. The bilayers of amphipathic molecules formed betweenthe contacting droplets in the first region may, for instance, comprisesaid membrane protein. Further, the bilayers of amphipathic moleculesformed between the contacting droplets in the second region may notcomprise said membrane protein or may comprise a lower concentration ofsaid membrane protein than the bilayers of amphipathic molecules formedbetween the contacting droplets in the first region.

The membrane protein may be of any type. The membrane protein may forinstance be a membrane pump, channel and/or pore, to allow for precisecontrol over the exchange of material, and electrical communication,between (i) individual droplets within the assembly and (ii) the dropletassembly and an external solution. The membrane protein could forinstance be an αHL pore. However, any suitable membrane protein can beused including one from the two major classes, that is, β-barrels orα-helical bundles. Besides a protein pore or channel, further possiblemembrane proteins include, but not exclusively, a receptor, atransporter or a protein which effects cell recognition or acell-to-cell interaction. The channel can be a voltage-gated ionchannel, a light-sensitive channel such as bacteriorhodopsin, aligand-gated channel or a mechano-sensitive channel.

Other suitable membrane protein include, but are not limited to,bacterial peptides and ionophores. The membrane protein may,alternatively, be an engineered membrane protein or synthetic membraneprotein. The engineered membrane protein may, for instance, be agenetically engineered protein, or a covalent or non-covalent chemicallyengineering protein. The synthetic membrane protein may, for instance,be a peptide or an organic molecule.

Typically, the membrane protein is an α-hemolysin (αHL) pore.

As discussed above, a droplet assembly can be made to fold as aconsequence of osmosis. The folding process may bring dropletspreviously not in contact with each other into contact with each other.The folding process may, for instance, result in the formation of newbilayers of amphipathic molecules.

In some embodiments, in the process of the invention, the aqueous mediumof the droplets dispensed into the hydrophobic medium in said first setof dispensing steps has a first osmolarity, and the aqueous medium ofthe droplets dispensed into the hydrophobic medium in said second set ofdispensing steps has a second osmolarity, wherein the first osmolarityis greater than the second osmolarity; and droplets in the second regionare disposed adjacent to droplets in the first region, so that dropletsin the first region contact droplets in the second region to formbilayers of said amphipathic molecules as interfaces between thecontacting droplets.

Typically, the aqueous medium of the droplets dispensed into thehydrophobic medium in said first set of dispensing steps is an aqueoussolution of a salt, which aqueous solution has said first osmolarity,and the aqueous medium of the droplets dispensed into the hydrophobicmedium in said second set of dispensing steps is an aqueous solution ofa salt, which aqueous solution has said second osmolarity. In someembodiments, the aqueous medium of the droplets dispensed into thehydrophobic medium in said first set of dispensing steps is an aqueoussolution of a first salt, which aqueous solution has said firstosmolarity, and the aqueous medium of the droplets dispensed into thehydrophobic medium in said second set of dispensing steps is an aqueoussolution of a second salt, which aqueous solution has said secondosmolarity. The first and second salt may, for instance, be differentsalts or the same salts. The first salt may, for instance, be achloride, such as potassium chloride, and the second salt may be acarbonate, such as potassium carbonate.

The ratio of the osmolarity of the first aqueous medium to theosmolarity of the second aqueous medium may be from 2:1 to 50:1, and ispreferably from 5:1 to 20:1. In some embodiments, the ratio of theosmolarity of the first aqueous medium to the osmolarity of the secondaqueous medium is from 5:1 to 15:1.

The extent to which a droplet assembly folds is typically determined bythe osmolarity ratio and the geometry of the droplet network: for agiven geometry. For instance, a network with a higher osmolarity ratiobetween the two droplet types may fold more rapidly than a network witha lower osmolarity ratio between the two droplet types.

The rate at which a droplet assembly folds it usually determined by thedifference in osmolarities. Typically, the folding rate is proportionalto the difference in osmolarities of the two droplets. For example, fora given ratio of osmolarities such as 10:1, a network in which theosmolyte concentrations are 1 M and 100 mM will fold approximately tentimes more quickly than one in which the concentrations are 100 mM and10 mM.

The rate of folding of the droplet assembly and the extend of foldingmay therefore be tuned.

The ability to tune the rate at which the droplet networks fold isimportant for the printing process. If the time taken to print a networkis comparable to the time over which it folds, then parts of the networkwill move during printing and the object will therefore be printedincorrectly. In some embodiments, a large concentration ratio (toachieve large deformations) but a small concentration difference areused. This makes the folding time significantly slower than the printingtime.

The rate of folding is also determined by the size of the droplets: fora given geometry, a network composed of larger droplets will usuallyfold more slowly. The folding rate is typically inversely proportionalto the droplet diameter.

In some embodiments, the aqueous medium of the droplets dispensed intothe hydrophobic medium in said first set of dispensing steps is anaqueous solution of a salt, which salt has a first concentration in theaqueous solution, and the aqueous medium of the droplets dispensed intothe hydrophobic medium in said second set of dispensing steps is anaqueous solution of the same salt, which salt has a second concentrationin the aqueous solution, wherein the first concentration is greater thanthe second concentration.

The ratio of the concentration of the salt or buffer in the firstaqueous medium to the concentration of the salt or buffer in the secondaqueous medium may be from 2:1 to 50:1, and is preferably from 5:1 to20:1.

Typically, the concentration of the salt or buffer in the first aqueousmedium is from 100 mM to 1,000 mM and the concentration of the salt orbuffer in the second aqueous medium is from 0.1 mM to 100 mM. Typically,the concentration of the salt or buffer in the first aqueous medium isfrom 150 mM to 750 mM and the concentration of the salt or buffer in thesecond aqueous medium is from 0.5 mM to 75 mM. More typically, theconcentration of the salt or buffer in the first aqueous medium is from150 mM to 500 mM and the concentration of the salt or buffer in thesecond aqueous medium is from 5 mM to 25 mM. For instance, theconcentration of the salt or buffer in the first aqueous medium may beabout 250 mM and the concentration of the salt or buffer in the secondaqueous medium may be about 25 mM.

Usually, the concentration of the salt or buffer in the first aqueousmedium and the concentration of the salt or buffer in the second aqueousmedium is a concentration of an alkali metal halide salt, such aspotassium chloride. For instance, the buffer solution may for instancecomprise Tris-HCl and/or KCl.

The process may, for instance, further comprises allowing water totransfer from the second region to the first region, to causedeformation of the droplet assembly. Typically, the transfer causes thefirst and second regions to curve or fold.

The deformation may, for instance, result in droplets previously not incontact with each other into, coming into contact with each other. Thedeformation may, for instance, result in the formation of new bilayersof amphipathic molecules.

The transfer typically occurs from one droplet to another, for instance,through the bilayer between contacting droplets. The transfer may, forinstance, occur from at least one droplet in the second region to atleast one droplet in the first region.

Typically, the first and second regions of droplets respectivelycomprise first and second layers of droplets are defined hereinabove,wherein the first and second layers are substantially rectangular andthe transfer causes the first and second layers to fold into asubstantially ring-shaped structure; the first layer is substantiallyrectangular and the second layer comprises parallel strips of droplets,and the transfer causes the first and second layers to fold into asubstantially cylindrical structure; or the first and second layerscomprise petal-shaped regions, and said transfer causes saidpetal-shaped regions to fold inwards and join to form a hollow dropletassembly.

More typically, the first and second regions of droplets respectivelycomprise first and second layers of droplets as defined hereinabove,wherein the first and second layers are substantially rectangular andthe transfer causes the first and second layers to fold into asubstantially ring-shaped structure; or the first and second layerscomprise petal-shaped regions, and said transfer causes saidpetal-shaped regions to fold inwards and join to form a hollow dropletassembly.

When the first layer is substantially rectangular and the second layercomprises parallel strips of droplets, the formerly straight parallelstrips typically form a ring (or rings) inside of the cylinder.

In some embodiments, the first and second regions of dropletsrespectively comprise first and second layers of droplets as definedhereinabove wherein the first and second layers comprise planarflower-shaped regions, and said transfer causes said planarflower-shaped regions to fold inwards and join to form a hollow dropletassembly. Typically, the hollow droplet assembly formed is spheroidal.

Usually, the first region comprises the first layer of droplets asdefined hereinabove and at least one other layer of droplets.

Typically, the second region comprises the first layer of droplets asdefined hereinabove and at least one other layer of droplets.

The term hollow refers to a volume within the droplet assembly that doesnot comprise a droplet. That volume may comprise a material orsubstance, for instance a therapeutic agent, such as a prodrug, or adiagnostic agent, such as a contrast agent, or an enzyme. In someembodiments, the volume comprises a living cell (or living cells), forexample for use in tissue engineering. The volume may comprise aninorganic compound or material.

The hollow droplet assembly may, for instance, be substantiallyspherical.

The droplet assembly produced may, for instance, comprise a volumewithin the assembly that does not comprise any droplets.

In some embodiments, in the process of the invention, the droplet mediumis an aqueous medium and the bulk medium is a hydrophobic medium and theapparatus comprises a first droplet generator and a second dropletgenerator, and the plurality of dispensing steps comprises: at least onedispensing step which comprises dispensing a droplet of a first aqueousmedium from the first droplet generator into the hydrophobic medium, inthe presence of amphipathic molecules, and thereby forming in thehydrophobic medium a said droplet which comprises (i) said first aqueousmedium and (ii) an outer layer of amphipathic molecules around thesurface of the aqueous medium; and at least one dispensing step whichcomprises dispensing a droplet of a second aqueous medium from thesecond droplet generator into the hydrophobic medium, in the presence ofamphipathic molecules, and thereby forming in the hydrophobic medium asaid droplet which comprises (i) said second aqueous medium and (ii) anouter layer of amphipathic molecules around the surface of the aqueousmedium, wherein the first aqueous medium and the second aqueous mediumare the same or different.

For instance, the aqueous medium may be pure water, or an aqueous buffersolution, or an aqueous solution of one or more salts, or an aqueoussolution comprising agarose and water. Usually, the buffer solutioncomprises Tris-HCl and/or KCl. Alternatively, the aqueous medium maycomprise a hydrogel.

Typically, the plurality of dispensing steps comprises a first pluralityof said dispensing steps which comprise dispensing a droplet of a firstaqueous medium from the first droplet generator into the hydrophobicmedium, and a second plurality of said dispensing steps which comprisedispensing a droplet of a second aqueous medium from the second dropletgenerator into the hydrophobic medium.

Usually, the first aqueous medium is different from the second aqueousmedium.

Typically, the first and second aqueous media have differentosmolarities, or the first and second aqueous media comprise differentconcentrations of a salt or buffer.

For instance, the ratio of the concentration of the salt or buffer inthe first aqueous medium to the concentration of the salt or buffer inthe second aqueous medium may be from 2:1 to 50:1, and is preferablyfrom 5:1 to 20:1.

Typically, the concentration of the salt or buffer in the first aqueousmedium is from 100 mM to 1,000 mM and the concentration of the salt orbuffer in the second aqueous medium is from 0.1 mM to 100 mM. Typically,the concentration of the salt or buffer in the first aqueous medium isfrom 150 mM to 750 mM and the concentration of the salt or buffer in thesecond aqueous medium is from 0.5 mM to 75 mM. More typically, theconcentration of the salt or buffer in the first aqueous medium is from150 mM to 500 mM and the concentration of the salt or buffer in thesecond aqueous medium is from 5 mM to 25 mM. For instance, theconcentration of the salt or buffer in the first aqueous medium may beabout 250 mM and the concentration of the salt or buffer in the secondaqueous medium be about 25 mM.

Usually, the concentration of the salt or buffer in the first aqueousmedium and the concentration of the salt or buffer in the second aqueousmedium is a concentration of an alkali metal halide salt, such aspotassium chloride. For instance, the buffer solution may compriseTris-HCl and/or KCl.

In some embodiments, the first aqueous medium comprises a membraneprotein and the second aqueous medium does not comprise said membraneprotein. Alternatively, the first and second aqueous media may havedifferent concentrations of a membrane protein.

In some embodiments the droplet or droplets of the first aqueous medium,which are dispensed by the first droplet generator, have a differentsize from the droplet or droplets of the second aqueous medium, whichare dispensed by the second droplet generator. In other embodiments, thedroplet or droplets of the first aqueous medium, which are dispensed bythe first droplet generator, have the same size as the droplet ordroplets of the second aqueous medium, which are dispensed by the seconddroplet generator.

As the skilled person will appreciate, when the droplet assemblyproduced by the process of the invention is a self-folding dropletassembly, the speed at which the assembly folds before the droplets ofthe assembly have all been dispensed, may affect the positioning ofindividual droplets. The folding of the assembly during the printingprocess should typically be minimised. Folding times may be lengthened,for instance, by reducing the difference in the concentration of thesalt or buffer in different droplets or by increasing the size of thedroplets. This is discussed further in the Examples, under SupplementaryDiscussion.

As mentioned for the apparatus of the invention, and as shown in FIG.10, the diameter of the droplet may be tuned by varying the amplitudeand duration of the voltage pulses. By varying these parameters, thedroplet diameter can be tuned to a suitable diameter. The diameter may,for instance, be tuned between about 10 and 200 μm.

When, for instance, the aqueous medium is dispensed from the or eachdroplet generator by the application of a voltage pulse to thepiezoelectric component, the voltage pulse may have a peak-to-peakamplitude of from 5 V to 100 V, for instance, of from 10 V to 80 V. Thepeak-to-peak amplitude may, for instance, be of from 20 V to 60 V.Typically, each pulse has a duration of from 10 to 1,500 μs, forinstance, of from 50 to 1,000 μs. More typically, each pulse has aduration of from 100 to 800 μs. Usually, the voltage pulse is a squarevoltage pulse.

The droplet generator may also be adapted to control the droplet size.The or each droplet generator is typically a droplet generator asdefined above for the apparatus of the invention. Typically, the outletof the or each droplet generator has a diameter of less than 500 μm, forinstance, of less than 250 μm. More typically, the outlet of the or eachdroplet generator has a diameter of less than 200 μm, for instance, ofless than 150 μm. For instance, the outlet of the or each dropletgenerator typically has a diameter of from 20 μm to 200 μm, for instancefrom 60 μm to 120 μm. The outlet of the or each droplet generator may,for instance, have a diameter of about 100 μm.

Usually, the or each droplet generator further comprises a capillaryattached to the chamber, wherein the tip of the capillary is saidoutlet. The tip of the capillary typically has a diameter of less than150 μm. For instance, the tip of the capillary may have a diameter offrom 20 μm to 200 μm, for instance from 60 μm to 120 μm. The tip of thecapillary may, for instance, have a diameter of about 100 μm.

As mentioned above, in the process of the invention, the droplet mediumis usually an aqueous medium and the bulk medium is usually ahydrophobic medium. In some such embodiments, said hydrophobic medium isa drop of a hydrophobic medium.

The drop of the hydrophobic medium may, for instance, be within a secondbulk medium which is an aqueous medium, wherein the container of theapparatus contains said second bulk medium which is an aqueous mediumand the drop of hydrophobic medium.

Usually, the drop of the hydrophobic medium further comprises aperipheral layer of amphipathic molecules around the surface of thedrop, as an interface between the drop and the second bulk medium whichis an aqueous medium. The amphipathic molecules may be the sameamphipathic molecules as the amphipathic molecules in the outer layer ofamphipathic molecules around the surface of the aqueous droplet mediumor they may be different amphipathic molecules. Typically, a bilayer ofamphipathic molecules is formed at the surface of the drop. There maytherefore be (i) droplet interface bilayers between droplets and (ii)bilayers between droplets and the surface of the drop. These bilayersallow communication (i) between droplets within the droplet assembly and(ii) between a droplet within the droplet assembly and the externalenvironment.

Typically, at least one droplet in the drop of the hydrophobic medium isin contact with the surface of the drop of the hydrophobic medium. Abilayer is typically formed at the point of contact. This separates thedroplet from the second bulk medium which is an aqueous medium.

In some embodiments, the droplet assembly is produced within the drop ofthe hydrophobic medium, to produce a droplet encapsulate, which dropletencapsulate comprises: said drop of the hydrophobic medium; saidperipheral layer of amphipathic molecules around the surface of thedrop; and said droplet assembly within the peripheral layer.

The drop may, for instance, be suspended in a second bulk medium whichis an aqueous medium. For instance, the drop may be suspended on aframe, such as a wire frame. Usually, the wire frame is coated with ahydrophobic coating.

Typically, the wire frame comprises a metal such as silver.

The hydrophobic coating on the wire frame usually comprises a polymersuch as poly(methyl methacrylate). The coating may cover all of the wireframe or it may cover part of the wire frame. The coating may, forinstance, cover the part of the wire frame that contacts the drop of ahydrophobic medium.

The wire frame may be any shape. For instance, the wire frame maycomprise a circular loop.

The droplet encapsulate may, for instance, be stable for at least aweek, for instance, at least two weeks. The stability of the dropletencapsulate means that they can functionalised and used for purposessuch as communication with its surroundings through membrane pores, andfor pH- or temperature-triggered release of contents.

Usually, when said hydrophobic medium is a drop of a hydrophobic medium,the process further comprises removing excess hydrophobic medium oncethe droplet assembly has been produced. Typically, the excesshydrophobic medium is removed using the or each droplet generator. Moretypically, the excess hydrophobic medium is removed by suction throughthe or each droplet generator. The removal of excess hydrophobic mediumtypically removes at least half of the hydrophobic medium, for instanceat least 75% of the hydrophobic medium.

In some embodiments, the excess hydrophobic medium may be removed byallowing a portion of it to dissolve into the bulk hydrophilic phase.For instance, a volatile solvent, such as a short chain hydrocarbon maybe added to the hydrophobic medium.

An encapsulate may comprise a droplet assembly comprising two or morecompartments (i.e. a multi-compartment droplet assembly). The dropletassembly may, for instance, comprise a first compartment and a secondcompartment. The first compartment within the droplet assembly maycommunicate with the second compartment via membrane proteins. The firstand/or second compartment may communicate with the external environmentvia membrane proteins. In principle, a droplet assembly may comprise alarge number of different compartments and architecturally definedstructures may thus be produced.

An encapsulate may comprise two or more droplet assemblies. Each dropletassembly may, for instance, be as defined herein.

In some embodiment individual droplet assemblies (such asthree-dimensional droplet assemblies) may be gelled together to form anew droplet assembly. In this way, individual droplet assemblies may beused as building bricks, for instance, to form complex structures. Thenew droplet assembly formed may, for instance, be part of anencapsulate.

The process of the invention may further comprise recovering saiddroplet assembly from the bulk medium. When the bulk medium is ahydrophobic medium, the process of the invention may further compriserecovering said droplet assembly from the hydrophobic medium.

In the embodiments of the process of the invention in which a dropletassembly is produced within a drop of the hydrophobic medium, to producea droplet encapsulate, the process of the invention may further compriserecovering said droplet encapsulate from the second bulk medium which isan aqueous medium.

Typically, in the process of the invention, the apparatus is as definedhereinabove for the apparatus of the invention.

The invention also relates to a droplet assembly which is obtainable bya process as defined hereinabove.

Further provided by the invention is a droplet assembly which comprisesa plurality of droplets, wherein each of said droplets comprises (i) anaqueous medium, and (ii) an outer layer of amphipathic molecules aroundthe surface of the aqueous medium, and wherein each of said dropletscontacts another of said droplets to form a bilayer of said amphipathicmolecules as an interface between the contacting droplets, wherein theplurality of droplets comprises a first region of said droplets and asecond region of said droplets, wherein each droplet in the first regioncontacts at least one other droplet in the first region to form abilayer of said amphipathic molecules as an interface between thecontacting droplets, and each droplet in the second region contacts atleast one other droplet in the second region to form a bilayer of saidamphipathic molecules as an interface between the contacting droplets,wherein the aqueous medium of the droplets in the first region has afirst osmolarity and the aqueous medium of the droplets in the secondregion has a second osmolarity, wherein the first osmolarity isdifferent from the second osmolarity.

As mentioned above, the difference in osmolarity between the firstosmolarity and the second osmolarity can cause a transfer of waterbetween droplets of different osmolarity and result in the dropletassembly self-folding. The droplet assembly may, for instance, bedesigned to fold in a predictable way. Such self-folding dropletassemblies may find application in areas such as tissue engineering. Bybuilding artificial tissues, many of the disadvantages of using livingcells are removed, for example, there would be no uncontrolledreplication or migration of cells and limited rejection of the materialin the body.

The aqueous medium may be any suitable aqueous medium. For instance, theaqueous medium may be pure water, or an aqueous buffer solution, or anaqueous solution of one or more salts. Alternatively, the aqueous mediummay comprise a hydrogel. When the aqueous medium may comprise ahydrogel, the aqueous medium may, for instance, comprise agarose andwater. The concentration of the agarose in water is typically less thanor equal to 10% w/v agarose. For instance, the concentration of theagarose in said water may be from 0.25 to 5% w/v agarose. Hydrogelsother than agarose may also be used. For instance the aqueous medium maycomprise methylcellulose, polyethylene glycol diacrylate,polyacrylamide, matrigel, hyaluronan, polyethylene oxide, polyAMPS(poly(2-acrylamido-2-methyl-1-propanesulfonic acid)),polyvinylpyrrolidone, polyvinyl alcohol, sodium polyacrylate, acrylatepolymers or poly(N-isopropylacrylamide). Alternatively, the aqueousmedium body may comprise a silicone hydrogel or LB (Luria broth) agar.

One important property of the aqueous medium is pH and this can bevaried over a wide range. In some embodiments, for instance, the pH ofthe aqueous medium within the aqueous droplet or droplets may be in therange of from 5 to 9 (or for instance in the range of from 6 to 8)although higher and lower pH values are also possible. The aqueousmedium may therefore be an aqueous buffer solution. Any suitable buffercan be employed, depending on the desired pH. The buffer solution mayfor instance comprise Tris-HCl and/or KCl. In some embodiments the pH ofthe aqueous buffer solution is from 5 to 9, or for instance from 6 to 8.The nature and concentration of the solutes can be varied to vary theproperties of the solution.

The aqueous medium of each droplet in the droplet assembly may be thesame or different.

The amphipathic molecules may be any suitable amphipathic molecules.Typically, the amphipathic molecules are amphipathic molecules asdefined above for the apparatus of the invention.

The droplet assembly may, for instance, be disposed in a hydrophobicmedium. The hydrophobic medium may, for instance, be a hydrophobicmedium as defined herein for the apparatus of the invention.

Typically, the droplet assembly comprises at least 100 of said droplets,each of which comprises (i) an aqueous medium and (ii) an outer layer ofamphipathic molecules around the surface of the aqueous medium. Moretypically, the droplet assembly comprises at least 1,000 of saiddroplets, each of which comprises (i) an aqueous medium and (ii) anouter layer of amphipathic molecules around the surface of the aqueousmedium. Even more typically, the droplet assembly comprises at least10,000 of said droplets, each of which comprises (i) an aqueous mediumand (ii) an outer layer of amphipathic molecules around the surface ofthe aqueous medium.

Usually, the first region and/or the second region comprises at least100 droplets, for instance at least 500 droplets. In some embodiments,the first region and/or the second region comprises at least 1,000droplets, for instance at least 50,00 droplets.

The number of droplets in each of the first and second regions may bevery large, for instance, at least 100,000. The number of droplets ineach of the first and second regions may be at least 1,000,000, forinstance, at least 10,000,000. For instance, the number of droplets ineach of the first and second regions may be at least 1,000,000,000. Insome embodiments, the number of droplets in each of the first and secondregions may be at least 10,000,000,000, for instance at least50,000,000,000.

Usually, droplets in the second region are disposed adjacent to dropletsin the first region, so that droplets in the first region contactdroplets in the second region to form bilayers of said amphipathicmolecules as interfaces between the contacting droplets.

Typically some, but not all, of the droplets in the second region aredisposed adjacent to droplets in the first region. Thus, typically onlysome of the droplets in the first region contact droplets in the secondregion to form bilayers. This will often be the case if the first and/orsecond region is a three-dimensional structure made up of a plurality oflayers of droplets. In such cases, only droplets on the surface of thefirst region will be able to contact droplets in the second region.

Usually, the ratio of the first osmolarity to the second osmolarity isfrom 2:1 to 50:1, preferably from 5:1 to 20:1.

In some embodiments, the aqueous medium of the droplets in the firstregion is an aqueous solution of a salt, which salt has a firstconcentration in the aqueous solution, and the aqueous medium of thedroplets in the second region is an aqueous solution of the same salt,which salt has a second concentration in the aqueous solution, whereinthe first concentration is different from the second concentration. Anysuitable salt may be used, for instance an alkali metal halide, such aspotassium chloride. In other embodiments, the aqueous medium of thedroplets in the first region is an aqueous solution of a first salt,which salt has a first concentration in the aqueous solution, and theaqueous medium of the droplets in the second region is an aqueoussolution of a second salt, which salt has a second concentration in theaqueous solution, wherein the first concentration is different from thesecond concentration. The first and second salt may be different salts.The first salt may, for instance, be a chloride, such as potassiumchloride, and the second salt may be a carbonate, such as potassiumcarbonate.

Typically, the ratio of the first concentration to the secondconcentration is from 2:1 to 50:1, preferably from 5:1 to 20:1.

In some embodiments, the concentration of the salt or buffer in thefirst aqueous medium is from 100 mM to 1,000 mM and the concentration ofthe salt or buffer in the second aqueous medium is from 0.1 mM to 100mM. Typically, the concentration of the salt or buffer in the firstaqueous medium is from 150 mM to 750 mM and the concentration of thesalt or buffer in the second aqueous medium is from 0.5 mM to 75 mM.More typically, the concentration of the salt or buffer in the firstaqueous medium is from 150 mM to 500 mM and the concentration of thesalt or buffer in the second aqueous medium is from 5 mM to 25 mM. Forinstance, the concentration of the salt or buffer in the first aqueousmedium may be about 250 mM and the concentration of the salt or bufferin the second aqueous medium may be about 25 mM.

Usually, the concentration of the salt or buffer in the first aqueousmedium and the concentration of the salt or buffer in the second aqueousmedium is a concentration of an alkali metal halide salt, such aspotassium chloride. For instance, the buffer solution may compriseTris-HCl and/or KCl.

In the droplet assembly of the invention, typically, the droplets in thefirst region are arranged in a row, a plurality of rows, a layer or aplurality of layers, and the droplets in the second region are arrangedin a row, a plurality of rows, a layer or a plurality of layers.

The first and second regions may be first and second rows as definedabove for the process of the invention. Alternatively, the first andsecond regions may be first and second layers as defined above for theprocess of the invention.

For instance, the first region of droplets may comprise a row ofdroplets, part of a row of droplets, a plurality of rows of droplets,part of a plurality of rows of droplets, a layer of droplets, part of alayer of droplets, a plurality of layers of droplets, or part of aplurality of layers of droplets; and/or the second region of dropletsmay comprise a row of droplets, part of a row of droplets, a pluralityof rows of droplets, part of a plurality of rows of droplets, a layer ofdroplets, part of a layer of droplets, a plurality of layers ofdroplets, or part of a plurality of layers of droplets.

In some embodiments, the first region of droplets forms a pathway ofdroplets through the second region of droplets. This is illustrated inFIGS. 12A, 12B, 12D.

In some embodiments, the bilayers of amphipathic molecules formedbetween the contacting droplets in the first region further comprisesaid membrane protein.

In some embodiments, the bilayers of amphipathic molecules formedbetween contacting droplets in the second region do not comprise saidmembrane protein.

In other embodiments, the aqueous medium of the droplets dispensed intothe hydrophobic medium in said first set of dispensing steps comprises ahigher concentration of a membrane protein than the aqueous medium ofthe droplets dispensed into the hydrophobic medium in said second set ofdispensing steps. The bilayers of amphipathic molecules formed betweenthe contacting droplets in the first region may, for instance, comprisesaid membrane protein. Further, the bilayers of amphipathic moleculesformed between the contacting droplets in the second region may notcomprise said membrane protein or may comprise a lower concentration ofsaid membrane protein than the bilayers of amphipathic molecules formedbetween the contacting droplets in the first region.

The membrane protein may for instance be a membrane pump, channel and/orpore, to allow for precise control over the exchange of material, andelectrical communication, between (i) individual droplets within theassembly and (ii) the droplet assembly and an external solution. Themembrane protein could for instance be an αHL pore. However, anysuitable membrane protein can be used including one from the two majorclasses, that is, β-barrels or α-helical bundles. Besides a protein poreor channel, further possible membrane proteins include, but notexclusively, a receptor, a transporter or a protein which effects cellrecognition or a cell-to-cell interaction. The channel can be avoltage-gated ion channel, a light-sensitive channel such asbacteriorhodopsin, a ligand-gated channel or a mechano-sensitivechannel.

Other suitable membrane protein include, but are not limited to,bacterial peptides and ionophores. The membrane protein may,alternatively, be an engineered membrane protein or synthetic membraneprotein. The engineered membrane protein may, for instance, be agenetically engineered protein, or a covalent or non-covalent chemicallyengineering protein. The synthetic membrane protein may, for instance,be a peptide or an organic molecule.

Typically, the membrane protein is an α-hemolysin (αHL) pore.

As discussed above, a droplet assembly may fold as a consequence ofosmosis.

In some embodiments, the droplets in the first region are arranged in alayer or a plurality of layers, and the droplets in the second regionare arranged in a layer or a plurality of layers, wherein droplets inthe second layer or plurality of layers are disposed adjacent todroplets in the first layer or plurality of layers, so that droplets inthe first layer contact droplets in the second layer to form bilayers ofsaid amphipathic molecules as interfaces between the contactingdroplets.

In some embodiments, for instance, the droplets in the first region arearranged in a layer or a plurality of layers, and the droplets in thesecond region are arranged in a layer or a plurality of layers, whereindroplets in the second layer or plurality of layers are disposed ondroplets in the first layer or plurality of layers, so that droplets inthe first layer contact droplets in the second layer to form bilayers ofsaid amphipathic molecules as interfaces between the contactingdroplets.

The first and second layers or pluralities of layers may, for instance,comprise petal-shaped regions.

Usually, the petal-shaped regions are capable of folding inwards andjoining to form a hollow droplet assembly. The hollow droplet assemblymay be spheroidal.

In one embodiment, the first and second layers or pluralities of layerscomprise flower-shaped regions. The flower-shaped regions may, forinstance, be capable of folding inwards and joining to form a hollowdroplet assembly. The hollow droplet assembly may be spheroidal.

The hollow droplet assembly may be substantially spherical.

As mentioned above, the term hollow refers to a volume within thedroplet assembly that does not comprise a droplet. That volume maycomprise a material or substance, for instance a therapeutic agent, suchas a prodrug, or a diagnostic agent, such as a contrast agent, or anenzyme. The volume may comprise a living cell, or living cells. Thevolume may comprise an inorganic compound or material.

The hollow droplet assembly may, for instance, be substantiallyspherical.

The droplet assembly produced may, for instance, comprise a volumewithin the assembly that does not comprise any droplets.

The invention also relates to a droplet assembly which comprises aplurality of droplets, wherein each of said droplets comprises (i) anaqueous medium, and (ii) an outer layer of amphipathic molecules aroundthe surface of the aqueous medium, and wherein each of said dropletscontacts another of said droplets to form a bilayer of said amphipathicmolecules as an interface between the contacting droplets, wherein theplurality of droplets defines a shell around a volume within the dropletassembly that does not comprise said droplets.

The aqueous medium may be any suitable aqueous medium. For instance, theaqueous medium may be pure water, or an aqueous buffer solution, or anaqueous solution of one or more salts. Alternatively, the aqueous mediummay comprise a hydrogel. When the aqueous medium may comprise ahydrogel, the aqueous medium may, for instance, comprise agarose andwater. The concentration of the agarose in water is typically less thanor equal to 10% w/v agarose. For instance, the concentration of theagarose in said water may be from 0.25 to 5% w/v agarose. Hydrogelsother than agarose may also be used. For instance the aqueous medium maycomprise methylcellulose, polyethylene glycol diacrylate,polyacrylamide, matrigel, hyaluronan, polyethylene oxide, polyAMPS(poly(2-acrylamido-2-methyl-1-propanesulfonic acid)),polyvinylpyrrolidone, polyvinyl alcohol, sodium polyacrylate, acrylatepolymers or poly(N-isopropylacrylamide). Alternatively, the aqueousmedium body may comprise a silicone hydrogel or LB (Luria broth) agar.

One important property of the aqueous medium is pH and this can bevaried over a wide range. In some embodiments, for instance, the pH ofthe aqueous medium within the aqueous droplet or droplets may be in therange of from 5 to 9 (or for instance in the range of from 6 to 8)although higher and lower pH values are also possible. The aqueousmedium may therefore be an aqueous buffer solution. Any suitable buffercan be employed, depending on the desired pH. The buffer solution mayfor instance comprise Tris-HCl and/or KCl. In some embodiments the pH ofthe aqueous buffer solution is from 5 to 9, or for instance from 6 to 8.The nature and concentration of the solutes can be varied to vary theproperties of the solution.

The aqueous medium of each droplet in the droplet assembly may be thesame or different.

The amphipathic molecules may be any suitable amphipathic molecules.Typically, the amphipathic molecules are amphipathic molecules asdefined above for the apparatus of the invention.

The droplet assembly may, for instance, be disposed in a hydrophobicmedium. The hydrophobic medium may, for instance, be a hydrophobicmedium as defined herein for the apparatus of the invention.

Typically, the droplet assembly comprises at least 100 of said droplets,each of which comprises (i) an aqueous medium and (ii) an outer layer ofamphipathic molecules around the surface of the aqueous medium. Moretypically, the droplet assembly comprises at least 1,000 of saiddroplets, each of which comprises (i) an aqueous medium and (ii) anouter layer of amphipathic molecules around the surface of the aqueousmedium. Even more typically, the droplet assembly comprises at least10,000 of said droplets, each of which comprises (i) an aqueous mediumand (ii) an outer layer of amphipathic molecules around the surface ofthe aqueous medium.

The volume that does not comprise any droplets may be completelyenclosed by the shell, or it may be partially exposed.

The volume within the droplet assembly may comprise a bioactive agent.For instance it may comprise a therapeutic, such as a prodrug, and/or adiagnostic agent, such as a contrast agent, or an enzyme. The volume maycomprise a living cell, or living cells.

Typically, the shell defined by said droplets is a curved structure.

Usually, the shell defined by said droplets is substantiallycylindrical, substantially ring-shaped, substantially spherical, orsubstantially hemispherical.

In some embodiments, the shell defined by said droplets encloses saidvolume within the droplet assembly.

The shell defined by said droplets may, for instance, be substantiallyspherical.

Typically, in the droplet assembly of the invention, the number of saiddroplets in the plurality of droplets is at least 100, for instance, atleast 500. In some embodiments, the number of said droplets in theplurality of droplets is at least 1000, for instance, at least 5000. Forinstance, number of said droplets in the plurality of droplets may be atleast 10000 or at least 30000. For instance, the number of said dropletsin the plurality of droplets may be about 35000.

The number of droplets in the plurality of droplets can in principle bevery high, for instance of the order of millions. Such networks, whichcan in principle comprise millions of droplets, may, for instance, beuseful for preparing prototissue (i.e. an aggregate of protocells). Insome embodiments, therefore, the integer n may be as high as severalmillion, for instance up to about 10,000,000, or for instance up toabout 5,000,000.

The invention also relates to the use of a droplet assembly as definedherein in synthetic biology. For instance, use in preparing a protocellor an aggregate of protocells.

The invention also relates to the use of a droplet assembly as definedherein in tissue engineering. The droplet assembly may, for instance, beused to augment or replace failing tissues or organs. A droplet, ordroplets, of the droplet assembly may comprise living cells. Forinstance, the cells may be allowed to grow inside the droplet afterprinting and/or to break down the bilayers between droplets afterprinting.

Also provided by the invention is the use of a droplet assembly asdefined herein for the droplet assembly of the invention as adrug-delivery vehicle.

The invention also provides the use of a droplet assembly as definedherein for the droplet assembly of the invention in material science andengineering.

Further provided by the invention is the use of a droplet assembly asdefined herein for the droplet assembly of the invention as a templatefor the patterning of a solid material. The solid material may, forinstance, be used in electronics, optics, photonics, or other materialscience applications. A droplet, or droplets, may, for instance,comprise inorganic materials that could diffuse between specificdroplets. The inorganic materials may then react to form inorganicsolids such as cadmium sulphide.

The present invention is further illustrated in the Examples whichfollow:

EXAMPLES

General Methods

The lipid in all the Examples discussed below was1,2-diphytanoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids) at0.2-0.5 mg ml⁻¹. The oil was a 1:1 (v/v) mixture of hexadecane andsilicone oil AR 20 (both from Sigma Aldrich). The buffer in allexperiments was 25 mM Tris-HCl, 1 M KCl, 100 μM EDTA, pH 8.0, except inthe folding experiments where this solution was diluted to obtain thesalt concentrations given in the brief description of the figuressection for to FIGS. 15A-15E. The dyes used were xylene cyanol FF,orange G and pyranine (all from Sigma-Aldrich), at the concentrationsgiven below, under Supplementary Methods.

Each droplet generator consisted of a piezoelectric transducer(7BB-20-6L0, Murata) affixed with epoxy adhesive onto a micromachinedpoly(methyl methacrylate) (PMMA) chamber (FIG. 3 and SupplementaryMethods). The nozzle was formed from a glass capillary (SupplementaryMethods) and fitted onto the chamber using a silicone rubber adapter(Drummond). The containers used for printing in bulk oil and bulkaqueous solution are described below, under Supplementary Methods.

The micromanipulator (PatchStar, Scientifica) was controlled by acomputer. The same computer controlled the two droplet generatorsthrough a single Arduino Uno microcontroller board (SmartProjects),which through a digital-to-analog converter (AD5504, Analog Devices) andoperational amplifier (OPA551, Texas Instruments) created an outputvoltage in the range −30 V to +30 V. The output voltage was appliedthrough a relay (NRP-04, NCR) to one of the two transducers at a time(see also Supplementary Methods). Voltage pulses typically had aduration of 100-800 μs and a peak-to-peak amplitude of 20-60 V. Thecomputer programs used to print networks in bulk oil and bulk aqueoussolution are described below, under Supplementary Methods.

Heptameric αHL used in the electrical pathway experiments was preparedas described (Maglia, G. et al., Nano Lett. 9, 3831-3836 (2009)), andadded to aqueous droplets at 100-fold dilution. The Ag/AgCl electrodeswere prepared by treating 100-μm diameter silver wire (Sigma Aldrich)with 25% sodium hypochlorite solution for >1 h. Currents were measuredwith the electrodes by using a patch-clamp amplifier (Axopatch 200B,Axon Instruments) and a 16-bit digitizer (1322A, Molecular Devices).Signals were processed with a 5 kHz low-pass Bessel filter and acquiredat 20 kHz.

Results Example 1 Droplet Generator

The automated production of droplet networks requires a reliable sourceof droplets of controlled size. The inventors built a piezoelectricdroplet generator that employs a tapered glass capillary as a nozzle(see General Methods below). The device was filled with aqueous bufferand the nozzle tip immersed in a solution of lipids in oil. The diameterof droplets ejected into the oil by applying a voltage to thepiezoelectric element (Supplementary Methods) could be tuned to between−10 μm and 200 μm by varying the amplitude and duration of the voltagepulse (FIG. 10, Supplementary Discussion).

Example 2 Printing of a Network of Droplets Using Two Droplet Generators

Networks of heterologous droplets were printed by using two dropletgenerators. The two nozzles were placed close together, and immersed ina bath of lipid-containing oil mounted on a motorized micromanipulator(FIG. 2A). The droplet generators and manipulator were controlled by acomputer. A program was written that allowed designed three-dimensionalnetworks to be printed automatically (Supplementary Methods). A networkis defined by the user as a series of images that represent horizontalcross-sections one droplet thick. Based on these images, the programsynchronizes the motion of the oil bath with the ejection of dropletsfrom the two nozzles to build the network up in horizontal layers (FIG.2B, Supplementary Methods).

Example 3 Printing of Millimetre-Sized Networks

In printing droplet networks, the inventors encountered challenges thatdo not arise in most two- and three-dimensional printing technologies(see also Supplementary Discussion). These are due to the relativelylong time required for droplets to acquire a lipid monolayer, sink totheir intended position, and form bilayers with other droplets. The timerequired for each of these processes is on the order of one second.Droplets that have not adhered to the printing substrate or formedbilayers with the growing network may be displaced from their intendedpositions, both by the ejection of further droplets and by viscous dragproduced by the motion of the nozzles in the oil. Through theappropriate choice of fluids, nozzle geometry, droplet size and printingalgorithm (Supplementary Discussion, Supplementary Methods), theinventors have printed precisely defined networks several millimetres insize, comprised of up to ˜35,000 heterologous droplets of diameter ˜50μm ejected at a rate of ˜1 s⁻¹ (FIGS. 2C-F).

Printed droplet networks are self-supporting (see for example FIG. 2F),and a thermodynamic analysis of the system indicates that stablenetworks can be printed with at least several thousand layers. Thelattice of lipid bilayers also allows droplet networks to retain theirshape under gentle perturbations; each bilayer lends an effective springconstant of ˜4 mN/m to connected droplets, with a tensile strength of˜25 Pa. The inventors estimate that the Young modulus of the material isof the order of ˜100-200 Pa for the conditions of the experiments. Thisrange of stiffness overlaps with the elastic moduli of brain, fat andother soft tissues (Levental, I., Georges, P. C. and Janmey, P. A. SoftMatter 3, 299-306 (2007)).

Example 4 Printing Inside an Oil Droplet

The inventors have shown previously (Villar, G., et al, Nature Nanotech.6, 803-808 (2011)) that droplet networks can be stabilized in bulkaqueous solution by encapsulation within small drops of oil, forprospective applications in synthetic biology and medicine. Whereaspreviously the encapsulated networks were created manually and thereforewere limited in complexity, here the inventors demonstrate the printingof complex encapsulated networks. This was achieved by printing insidean oil drop suspended in aqueous solution (FIG. 11A) (SupplementaryMethods, Supplementary Discussion). Once printing is complete, excessoil can be removed by suction through one of the printing nozzles.Encapsulated printed networks (FIGS. 11B-D) were stable for at leastseveral weeks, and will therefore serve to expand the functionspreviously demonstrated with simple encapsulated networks, includingcommunication with the aqueous surroundings through membrane pores, andpH- or temperature-triggered release of contents (Villar, G., et al.,Nature Nanotech. 6, 803-808 (2011)).

Example 5 Droplet Assembly Comprising a Communication Pathway

Having demonstrated that droplet networks can be stabilized in bulkaqueous solution, the inventors aimed to further develop dropletnetworks as minimal analogues of functional tissue by printing networkswith membrane proteins in specific interface bilayers. To this end, theyprinted a network in which only the droplets along a defined pathwaycontained staphylococcal α-hemolysin (αHL) (FIGS. 12A, 12B). Theinventors determined whether protein pores inserted into bilayers alongthe pathway by electrical recording. To probe the network electricallyin a non-destructive way, a drop of buffer of diameter ˜500 μmcontaining αHL was manually pipetted onto each of two Ag/AgClelectrodes. The drops were then brought into contact with differentparts of the network, so that they formed bilayers with the droplets onthe network surface (FIG. 12A).

When the two large drops were placed on either end of the αHL-containingpathway (FIG. 12B), the inventors measured a stepwise increase in ioniccurrent under an applied potential (FIG. 12C), caused by the insertionof αHL pores into the new bilayers. After one of the drops was separatedand brought back into contact with the network away from theαHL-containing pathway (FIG. 12D), only transient currents were observed(FIG. 12E). When this drop was separated from the network again andreplaced in its original position, a stepwise increase in current wasagain observed (data not shown). Droplet networks in which no dropletscontained αHL showed negligible current flow, whereas the currentmeasured across droplet networks in which every droplet contained αHLwas similar to that shown in FIG. 12C (FIG. 13D).

To facilitate the interpretation of these results, a formalism for thecomputational simulation of the electrical behaviour of complex dropletnetworks was developed (see Supplementary Methods below). The electricalmodel was found to be consistent with the measured currents exemplifiedin FIGS. 12C, 12E, if most of the bilayers along the pathway containedseveral αHL pores, and the other bilayers in the network contained none(Supplementary Discussion), so that the pathway presented negligibleelectrical resistance compared to the rest of the network. The stepwiseincrease in current in FIG. 12C was therefore most likely caused by poreinsertions in the bilayers between the pathway droplets and the largedrops (Supplementary Discussion). The current spikes in FIG. 12Ecorrespond to pore insertions in the bilayers between the drop placedaway from the pathway and insulating droplets in the network(Supplementary Discussion). Each pore insertion in one of these bilayersreduces the resistance of that bilayer, which in turn increases the flowof ionic current through that bilayer. However, because each insulatingdroplet does not provide a route for ionic flow through its otherbilayers, a net ionic charge accumulates in the droplet. This capacitivecharging of the insulating layer of droplets produces a voltage thatacts to eliminate the flow of ionic current. The current is thereforemeasured as a transient spike

Based on these findings, the inventors have shown that droplet networkscan be printed with protein pores in specific bilayers, and so allow theflow of ionic current along a defined pathway under an appliedpotential. In enabling rapid electrical communication along a pathbetween two sites, the network presented here mimics the essentialfunction of a nerve, but not its mechanism of signal propagation. Moresophisticated networks with functional membrane channels or pores couldexhibit more complex behaviour (Maglia, G. et al., Nature Nanotech. 4,437-440 (2009)). For instance, a network might employ voltage-gated ionchannels to generate and transmit an action potential along its length,or light-sensitive channels such as bacteriorhodopsin to mimic thefunction of the retina (Holden, M. A., et al., J. Am. Chem. Soc. 129,8650-8655 (2007)).

Example 6 Folding Networks

A means to fold printed droplet networks into 3D forms that are notreadily obtained by printing alone was also investigated. Waterpermeates readily through droplet interface bilayers even in the absenceof protein channels or pores, with a permeability coefficient of 27±5 μms⁻¹ (mean±s.d., n=6) under the conditions of this study (FIGS. 16A and16B, Supplementary Methods), consistent with other permeabilitymeasurements of droplet interface bilayers (Dixit, S. S., et al.,Langmuir 28, 7442-7451 (2012) and Xu, J., et al., Adv. Mater. 22,120-127 (2010)) and other lipid bilayer systems (Boroske, E., et al.,Biophys. J. 34, 95-109 (1981)). Consequently, two droplets of higher andlower osmolarity joined by a DIB will respectively swell and shrinkuntil their osmolarities are equal (FIG. 15A). By extension, watertransfer between droplets in a network composed of droplets of differentosmolarities will cause spontaneous deformation of the network as longas adhesion between droplets is maintained (FIG. 15B). Severalprerequisites were found for droplet networks to fold in a predictableway (see also Supplementary Discussion). First, to prevent droplets frombeing printed onto incorrect positions in the network, the network mustfold slowly compared to the printing time. Second, the swelling andshrinking of the two droplet types can produce a length mismatch betweenregions of connected droplets, and the induced stress can cause thenetwork to buckle in an uncontrolled manner. This is analogous to thebuckling instability in tissues that grow at inhomogeneous rates, suchas certain leaves (Nath, U., et al., Science 299, 1404-1407 (2003) andSharon, E., et al., Phys. Rev. E 75 (2007)) and flower petals (Liang, H.Y. & Mahadevan, L., Proc. Natl. Acad. Sci. USA 108, 5516-5521 (2011)),as well as in some synthetic systems (Klein, Y., et al., Science 315,1116-1120 (2007) and Sharon, E., et al., Nature 419, 579-579 (2002)). Incertain cases, the stress may instead cause connected droplets toseparate and thereby prevent further folding of the network around thefracture zone.

These various problems can be solved through judicious choices ofprinting rate, salt concentrations and droplet size, and adjustments tothe network geometry (see Supplementary Discussion). The final geometryof the network is then determined in a controlled way by its initialgeometry, the distribution of the two types of droplets, and the ratioof their osmolarities. The inventors formulated a simple model thatallows them to qualitatively predict the folding behaviour of a givendroplet network (see Supplementary Methods below), and this model wasused to design droplet networks that folded successfully.

In one experiment, the inventors printed a network that comprised twostrips of droplets of different salt concentrations, connected alongtheir lengths (FIG. 15C). The network folded spontaneously in thehorizontal plane over ˜3 h, until droplets in opposing ends of thenetwork formed bilayers in a closed ring. They also programmed a networkto fold spontaneously out of the horizontal plane to attain a geometrythat would be difficult to print directly. A flower-shaped network withfour petals was printed, in which the lower layers had higher osmolaritythan the upper layers. The permeation of water from the upper into thelower layers induced a curvature that raised the petals and folded theminwards, in a manner that resembles the nastic movements exhibited bycertain plants (Forterre, Y., et al., Nature 433, 421-425 (2005) andSkotheim, J. M. & Mahadevan, L., Science 308, 1308-1310 (2005)). Thefolded network had a near-spherical geometry, with the originally upperlayer contained within a shell formed by the originally lower layer(FIGS. 15D, 15E). The evolution of the geometry of the network is ingood qualitative agreement with that of a simulated folding network withsimilar initial conditions (FIG. 15F).

Other materials exist that deform through non-uniform volume changes.For instance, the function of the bimetallic strip relies on aninhomogeneous thermal expansion coefficient. Also, hydrogel systems havebeen spatially patterned to suffer inhomogeneous changes in volume as afunction of solvent concentration or temperature (Kim, J., et al.,Science 335, 1201-1205 (2012) and Hu, Z. B., et al., Science 269,525-527 (1995)). Whereas these systems are driven by an externalstimulus, droplet networks fold as a result of water transfer entirelywithin the network, and therefore do not require an external drivingforce.

Supplementary Methods 1 Dye Concentrations

This section details the dye concentrations for each network shown inFIGS. 2D, 2F, 11B, 12B-E, 15C and 15D.

FIG. 2D: Dark grey droplets contain 1 mM xylene cyanol FF.

FIG. 2F: Dark grey droplets contain 1 mM xylene cyanol FF, and lightgrey droplets contain 10 mM orange G.

FIG. 11B: Light grey droplets contain 900 μM xylene cyanol FF and 100 μMpyranine, and dark grey droplets contain 10 mM orange G.

FIGS. 12B-E: Light grey droplets and large dark grey drops contain 10 mMpyranine, and other droplets contain 50 μM xylene cyanol FF.

FIG. 15C: Dark grey droplets contain 320 μM xylene cyanol FF, and lightgrey droplets contain 2.5 mM orange G.

FIG. 15D: Dark grey droplets contain 160 μM xylene cyanol FF, and lightgrey droplets contain 800 μM orange G.

2 Containers Networks Printed in Bulk Oil

The container for networks printed in bulk oil was a well micromachinedfrom poly(methyl methacrylate) with a glass observation window on oneside. The oil-filled volume was ˜15×10 mm horizontally and ˜5 mm deep.Spontaneously folding networks were printed directly on the bottomsurface of the well, while all other networks in bulk oil were printedon a piece of glass coverslip placed in the well.

Networks Printed in Bulk Aqueous Solution

The container for networks printed in bulk aqueous solution was apolystyrene cuvette, with a glass coverslip bottom in the case of thenetwork imaged by confocal microscopy.

Printing in bulk aqueous solution took place inside a drop of oil thatwas suspended on a wire frame, which was made as follows. Poly(methylmethacrylate) shavings were dissolved in chloroform at 100 mg ml⁻¹. Oneend of a 100-μm diameter silver wire (Sigma-Aldrich) was dipped in thissolution up to five times, so that it acquired a thin hydrophobiccoating. The coated end of the wire was then shaped into a loop usingtweezers, such that the loop was the only length of the wire coated withpolymer. The uncoated end was attached to the polystyrene containerusing epoxy adhesive.

To insert the printing nozzles into the oil drop, the nozzles first hadto pass through the bulk aqueous phase. To prevent leakage of the nozzlecontents into the bulk aqueous solution, a plug of the same oil mixtureused for the oil drop was sucked into the tip of each nozzle, byapplying suction at the inlet of each droplet generator with amicropipette. Once the nozzle tips had been placed inside the oil dropin aqueous solution, the oil plugs were expelled by applying positivepressure at each inlet using a micropipette.

3 Droplet Generator Nozzle

The printing nozzle for each droplet generator was formed from a glasscapillary (Drummond) with external and internal diameters of 1.4 mm and1.0 mm, respectively. The capillary was pulled (PC-10, Narishige), andits pulled end trimmed by gently passing another pulled capillary tipagainst it (Oesterle, A. P-1000 & P-97 Pipette Cookbook (rev. G). SutterInstrument Co., Novato (2011)) to give a flat-ended tip of diameterbetween ˜60 μm and 120 μm. The capillary was then bent by 900 over aflame, ˜15 mm from the pulled end. Finally, the capillary was trimmed˜35 mm from the pulled end (FIG. 3).

Filling

The chamber was filled with ˜400 μl of aqueous solution through theinlet on the top of the device by using a micropipette with agel-loading tip. The nozzle spontaneously filled with this solutionthrough capillary action.

The volume of aqueous solution required for printing could be reduced to˜5 μl, which minimized the wastage of solution in the experiments thatemployed αHL. To do this, the droplet generator was first filled withwater. The nozzle of the generator was then immersed in a well filledwith hexadecane, and suction was applied at the inlet of the generatorby using a micropipette so that ˜5 μl of hexadecane was drawn into thenozzle. The nozzle was then immersed into another well that containedthe aqueous sample, and suction applied to load a similar volume of theaqueous solution. The hexadecane formed a plug within the nozzle thatprevented the aqueous sample in the nozzle tip from mixing with thelarger volume of water. For significantly smaller loadings of theaqueous sample, the size of ejected droplets was found to vary with thevolume of aqueous sample remaining in the nozzle.

4 Driving Electronics for Droplet Generators

The electronic circuit built to drive the droplet generators is shownschematically in FIG. 4. The circuit interprets instructions from acomputer to produce a square voltage pulse of specified duration andamplitude, and applies this voltage to the piezoelectric transducer, orpiezo, in either of two droplet generators. This section describes howthe circuit generates the voltage pulse.

Piezo Selection

First, the computer sends a serial message that represents the desiredpiezo to an Arduino microcontroller board. The microcontrollerinterprets this message, and activates a relay through a transistor suchthat the desired piezo will receive the voltage output of the circuit.Once a piezo is selected in this way, it is held at the maximum negativevoltage of −30 V. The piezo terminal that is connected to the voltageoutput was chosen such that a negative voltage produces compression ofthe piezo.

Voltage Output

The computer then sends a serial message to the microcontroller thatrepresents the instruction to generate a voltage pulse. The duration andamplitude of the pulse can be either specified in the serial message orpreviously programmed into the microcontroller. The microcontrollerinterprets this message, and writes a value encoding the amplitude ofthe voltage pulse to the digital-to-analog converter (DAC) through aserial peripheral interface (SPI) bus. The DAC outputs a correspondingvoltage between 0 V and +30 V with 12-bit resolution. The DAC outputenters an operational amplifier circuit that acts as a current buffer,and also offsets and increases the output voltage range to −30 V to +30V. The output of this circuit is applied to the piezo previouslyselected by the relay. After the specified duration of the voltagepulse, the microcontroller again sets the piezo to the maximum negativevoltage.

5 Graphical User Interface

A graphical user interface that enables real-time control of the dropletprinter was written in the PRO-CESSING programming language (FIG. 5).The interface affords precise control of the amplitude and duration ofthe voltage pulse applied to each droplet generator. Droplet ejectioncan be triggered on demand, with a user-defined number of droplets andtime delay between multiple droplets. These features allow the user todetermine quickly, for each generator, the conditions required for theproduction of droplets of a specific size. The interface also gives theuser direct control of the motorized micromanipulator, which was used todetermine the correct spacing between droplets of a given size, as wellas the relative displacement of the two nozzles. The latter wascompensated for in the printing software to prevent a systematicdisplacement between the two types of droplets. The interface can becontrolled through the computer keyboard or mouse.

6 Printing Algorithm

Droplet networks were printed according to an algorithm written in thePROCESSING programming language, and executed by the computer thatcontrolled the two droplet generators and the motorizedmicromanipulator. Described first is the general pattern in whichnetworks are printed, followed by a description of the printingalgorithm.

6.1 Printing Pattern Layers

The algorithm builds droplet networks by printing one horizontal layerat a time. The network to be printed is defined by a series of images,or maps, each of which represents one or more of the layers in thenetwork (FIG. 2C). The number of layers represented by each map isspecified by the user. Each pixel in a map may have one of threecolours. Depending on its colour, each pixel represents a droplet fromone the two droplet generators, or the absence of a droplet; these arerespectively referred to here as A, B, and empty pixels.

Passes

Each layer is printed in two passes: in the first pass only the Adroplets of the layer are printed, and in the second pass only the Bdroplets. Each pass is printed one row at a time, with each row parallelto the horizontal dimension x.

Goals

The path that the printing nozzles are instructed to follow in a givenpass is defined by an ordered set of coordinates, or goals, each ofwhich represents a location at which a droplet is to be ejected. Foreach layer, a goal is set for each pixel in the corresponding map. Thegoal order begins at the minimum x and y, increases left to right alongx in even-numbered rows, increases along y, and increases right to leftin odd-numbered rows (FIGS. 6A and 6B).

Margin

When the rectangular pattern in FIG. 6B was used to print networksdesigned to be cuboidal, the resulting networks often had sloped wallsand a convex upper surface (FIGS. 7A, 7B). Observation of the printingprocess revealed that these deformations arose as a consequence of thefinite time required for two droplets to form a bilayer after cominginto contact (Supplementary Discussion). Droplets ejected at theoutermost edges of the network therefore tended to roll down the outerwalls for some distance before incorporating into the network. Althoughdroplets in the first few layers sometimes rolled towards the centre ofthe network instead of away from it, this increased the probability thatdroplets subsequently printed at the same position would roll outwards.

The tendency of droplets to roll down the outer walls caused a depletionof droplets in the outermost parts of upper layers of the network, andan excess around the outermost parts of lower layers. In a few cases,however, networks printed in the same way did not exhibit the samedistortion (FIG. 7C). In these cases, the motion of the nozzles throughthe oil dragged droplets towards the centre of the network before thedroplets rolled down the sides.

It was found that the printing pattern could be modified slightly toconsiderably and reliably improve the accuracy with which networks wereprinted. The modification consisted of (i) the addition of an outermargin of one or two pixels to the network maps, and (ii) themodification of the printing algorithm so that in addition to the normalejection of the droplets in each row, a droplet was ejected anadditional n times at each of the marginal goals, where n˜2 or 3depending on the network printed. Although the first marginal dropletsin each layer typically rolled down the outer walls of the network for afew layers, the following marginal droplets in that layer tended toincorporate into the network on top of these. In this way, the printingalgorithm forms a barrier of marginal droplets that grows at the pace ofthe network, and prevents the internal droplets from rolling out oftheir intended boundary.

6.2 Algorithm: Initialization

The printing algorithm (FIG. 8) begins with an initialization phase thatis executed once, followed by a direction phase that persists untilprinting has completed. In the initialization stage, the user firstinputs the parameters shown in Table 1. Two of the parameters, the goalskipping threshold and the interface reset period, are explained indetail below. The program then loads into memory all the maps thatdefine the network to be printed. A path for the printing nozzles,encoded as a series of goals according to the pattern detailed above, isestablished for the first pass of the first layer. Finally, themanipulator is instructed to move to the first goal. Once that goal isreached, the program sets a lower speed for the subsequent motion of themanipulator.

Goal Skipping Threshold

In a given pass, in general not all goals represent droplets of thecolour corresponding to that pass; for example, in a layer that containsboth A and B droplets, the A pass would include goals for B droplets.This can cause the manipulator to spend a significant time in travellingto goals at which no droplets are produced. This could be solved bysimply skipping (that is, omitting from the printing path) all the goalsthat do not require droplets in the current pass. However, the motion ofthe nozzles through the oil can cause the displacement of droplets thathave been recently produced (Supplementary Discussion). Therefore,skipping every goal that does not correspond to the current pass wouldcause some droplets in the network to be displaced differently toothers, depending on their positions along the printing path.

The algorithm prevents this problem by allowing the manipulator to skipto the next goal that requires a droplet in the current pass only if nodroplets have been ejected at any of the previous n goals, where n isthe goal skipping threshold. The goal skipping threshold is chosen toallow recently-ejected droplets enough time to incorporate into thenetwork, and so be unaffected by the motion of the printing nozzles.

Interface Reset Period

If the droplet generator chamber was too high above the level of oilsolution in the well, the hydrostatic pressure from the aqueous solutioncaused the printing nozzle to spontaneously leak its contents into theoil. With the chamber ˜1 cm above the oil and a typical nozzle with tipdiameter ˜80 μm, this leakage took place in two stages: an initial phasein which the aqueous volume gradually grew out of the nozzle to form anapproximately hemispherical protrusion over several minutes, followed bya rapid phase in which the contents of the aqueous chamber emptied intothe oil over a few seconds.

During the slower initial phase of leakage, the ejection of a dropletreturned the aqueous-oil interface at the nozzle to its original planargeometry. The droplet generator that was used in given pass wastherefore prevented from leaking its contents, because it ejecteddroplets at the relatively high rate of ˜1 s⁻¹. However, because eachpass took up to several minutes, the generator not used in a given passcould reach the rapid phase of leakage and empty its contents.

It was found that the application of certain voltage waveforms to adroplet generator would return the aqueous-oil interface at its nozzleto a planar geometry without causing the ejection of a droplet. Thevoltage waveform used to reset the interface typically consisted ofthree square pulses, each of duration 40 μs and amplitude ˜12 V, with a20 ms interval between them. In each pass, this waveform was appliedevery n goals to the droplet generator not used in that pass, where n isthe interface reset period.

TABLE 1 Printing parameters. x and y are the two horizontal dimensions,and rows of droplets are printed along x. In each layer, droplets inalternate rows can be printed with a displacement along x to promote theformation of a regular close-packed arrangement. Similarly,displacements in x and y can be set for droplets in alternate layers.The parameters goal skipping threshold and interface reset period areexplained in the text. Parameter Typical value Spacing between dropletsin x and y 50 μm x-offset for alternate rows 25 μm x- and y-offsets foralternate layers 25 μm Manipulator speed during printing 200 μm s⁻¹Delay after ejecting each droplet 200 ms Delay after printing each row 2s Goal skipping threshold 4 Interface reset period 10

6.3 Algorithm: Direction

In the direction stage, the computer synchronizes the motion of the oilbath with the ejection of droplets from the two generators according tothe algorithm in FIG. 8. If the manipulator has reached its currentgoal, a droplet is ejected if the goal colour matches that in thecurrent pass. If the goal was the last in the current pass, the passnumber and layer number are updated. If the goal was the last in theentire network, the nozzles are raised out of the oil to prevent theirleakage. Otherwise, the manipulator is instructed to travel to the nextgoal.

7 Electrical Simulation of Complex Droplet Networks 7.1 Statement of theProblem

The inventors consider a set of N droplets, some of which are joinedpairwise by bilayers in a given configuration. The bilayers may havedifferent areas and contain any number of protein pores, and the bilayerareas and numbers of pores may vary with time. It was assumed that twoof the N droplets, labelled a and b, are impaled by electrodes andpoised at known voltages u_(a) and u_(b), respectively. The system ismodelled as in FIG. 9, with each bilayer and any pores it containsrepresented by a capacitor and resistor in parallel. Given a dropletnetwork of known connectivity, and bilayers of known conductance andcapacitance, the inventors wish to calculate the current measured by theelectrodes in droplets a and b.

7.2 Mathematical Formulation

The current flowing from droplet j, at electrical potential V_(j), intodroplet i, at electrical potential V_(j), is given by I_(ij)=I_(ij)^(r)+I_(ij) ^(c), where

I _(ij) ^(r) =g _(ij)(V _(i) −V _(j)),

I _(ij) ^(c) =c _(ij)({dot over (V)} _(i) −{dot over (V)} _(j)),  (1)

and g_(ij)=1/r_(ij) is the conductance between droplets i and j, andc_(ij) is the capacitance between droplets i and j. The net current intothe ith droplet is given by the sum of the contributions from alldroplets in the network:

I _(i)=Σ_(j≠i) I _(ij),  (2)

where the sum extends over the entire network and the inventors defineg_(ij)=0 and c_(ij)=0 if droplets i and j are not joined by a bilayer.Substituting Eqs. (1) into Eq. (2) gives

I _(i)=Σ_(j≠i)(g _(ij)(V _(i) −V _(j))+c _(ij)({dot over (V)} _(i) −{dotover (V)} _(j))),

which can be rearranged as

$\begin{matrix}{I_{i} = {{V_{i}{\sum\limits_{j \neq i}g_{ij}}} - {\sum\limits_{j \neq i}{g_{ij}V_{j}}} + {{\overset{.}{V}}_{i}{\sum\limits_{j \neq i}c_{ij}}} - {\sum\limits_{j \neq i}{c_{ij}{\overset{.}{V}}_{j}}}}} & (3)\end{matrix}$

At this point it is convenient to define the matrices G and C asfollows:

$G_{ij} = \left\{ {{\begin{matrix}{{- g_{ij}},} & {{i \neq j},} \\{{\sum_{k \neq i}g_{ik}},} & {i = {j.}}\end{matrix}C_{ij}} = \left\{ \begin{matrix}{{- c_{ij}},} & {{i \neq j},} \\{{\sum_{k \neq i}g_{ik}},} & {i = {j.}}\end{matrix} \right.} \right.$

With these matrices, Eq. (3) may be written succinctly as

{right arrow over (I)}=G{right arrow over (V)}+C{right arrow over ({dotover (V)})},  (4)

where {right arrow over (I)} and {right arrow over (V)} areN-dimensional vectors that respectively represent the current owinginto, and the voltage at, each droplet.

Finally, it was assumed that the droplets cannot act as sources or sinksof charge. The current flowing into each droplet is therefore zero, withthe exception of the two droplets that are impaled by electrodes, whichmust source and sink the same current I:

$\begin{matrix}{{I_{i}} = \left\{ \begin{matrix}{I,} & {{i \in \left\{ {a,b} \right\}},} \\{0,} & {{otherwise}.}\end{matrix} \right.} & (5)\end{matrix}$

7.3 Initial Conditions

It was assumed that the system begins in a steady state. Then {rightarrow over ({dot over (V)})}={right arrow over (0)}, so from Eq. (4) theinventors have:

G{right arrow over (V)}={right arrow over (I)}.  (6)

To solve for {right arrow over (V)}, it is recalled that the twoterminal droplets are voltage-clamped. It is therefore possible toeliminate the two equations corresponding to droplets a and b, and useEq. (5) to obtain:

{tilde over (G)}{right arrow over (V)}={right arrow over (0)},  (7)

Where {tilde over (G)} represents the matrix G without the two rowscorresponding to droplets a and b.

Eq. (7) represents N−2 equations in N variables. The two additionalequations required to solve this system express the known voltages ofthe terminal droplets: V_(a)=u_(a) and V_(b)=u_(b). The inventorsinclude these equations by constructing a further matrix {tilde over(G)}′, defined by replacing the rows in G that were removed to form{tilde over (G)} with the row vectors {right arrow over (x)} and {rightarrow over (y)}, respectively, defined by x_(i)=δ_(ia) and y_(i)=δ_(ib),where δ_(ij) is the Kronecker delta. {tilde over (G)}′ is thereforedefined as:

${\overset{\sim}{G}}_{ij}^{\prime} = \left\{ \begin{matrix}{{{\delta_{ai}\delta_{aj}} + {\delta_{bi}\delta_{bj}}},} & {i \in \left\{ {a,b} \right\}} \\{{- g_{ij}},} & {{i \notin {{\left\{ {a,b} \right\}\mspace{14mu}{and}\mspace{14mu} i} \neq j}},} \\{{\sum_{k \neq i}g_{ik}},} & {{i \notin {\left\{ {a,b} \right\}\mspace{14mu}{and}\mspace{14mu} i}} = {j.}}\end{matrix} \right.$

Multiplying {tilde over (G)}′ by {right arrow over (V)} then gives

{tilde over (G)}′{right arrow over (V)}={right arrow over (v)},

where v_(i)=u_(a)δ_(ia)+u_(b)δ_(ib). This system of N equations in Nvariables is easily solved computationally, and yields the initialvoltage of each droplet. The initial current is then foundstraightforwardly from Eq. (6).

7.4 Time Evolution

The time evolution of the system can be calculated in a manner similarto the initial conditions. The inventors again begin with Eq. (4), andremove the two rows of G and C that correspond to droplets a and b tocreate G and C, where C is defined analogously to G. Eq. (4) thenbecomes:

{tilde over (G)}{right arrow over (V)}+{tilde over (C)}{right arrow over({dot over (V)})}={right arrow over (0)}.

The two rows removed from {tilde over (G)} and {tilde over (C)} werereplaced with {right arrow over (x)} and {right arrow over (y)} tocreate {tilde over (G)}′ and {tilde over (C)}′, where:

${\overset{\sim}{C}}_{ij}^{\prime} = \left\{ \begin{matrix}{{{\delta_{ai}\delta_{aj}} + {\delta_{bi}\delta_{bj}}},} & {i \in \left\{ {a,b} \right\}} \\{{- c_{ij}},} & {{i \notin {{\left\{ {a,b} \right\}\mspace{14mu}{and}\mspace{14mu} i} \neq j}},} \\{{\sum_{k \neq i}c_{ik}},} & {{i \notin {\left\{ {a,b} \right\}\mspace{14mu}{and}\mspace{14mu} i}} = {j.}}\end{matrix} \right.$

Recalling that the voltages V_(a)=u_(a) and V_(b)=u_(b) are constant,the following is obtained:

{tilde over (G)}′{right arrow over (V)}+{tilde over (C)}′{right arrowover ({dot over (V)})}={right arrow over (v)}.  (8)

The evolution of {right arrow over (V)} through time was calculated asfollows using MATLAB's ode45 ordinary differential equation solver:

1. At each time point t, G(t) is updated to reflect any changes inconductance that took place since the last time point to simulate poreinsertions. It was assumed that there are no changes in bilayer sizesand that no new bilayers are formed, although these could be easilysimulated by also updating C(t) at this step.

2. {right arrow over (V)}(t) and the updated G(t) and C(t) are used inEq. (8) to calculate {right arrow over ({dot over (V)})}(t).

3. {right arrow over (V)}(t) and {right arrow over ({dot over (V)})}(t)are used to calculate the voltages at the next time point, {right arrowover (V)} (t+δt). The interval δt is determined by the ode45 solver.

Once {right arrow over (V)}(t) has been calculated for the time periodof interest, the current I(t) can be found straightforwardly from Eq.(4).

8 Water Permeability of Droplet Interface Bilayers

A pair of droplets labelled 1 and 2, joined by a bilayer of area A, wereconsidered. Let the initial volumes of the droplets be V_(i) and V₂, andtheir salt concentrations at time t be C₁(t) and C₂(t). The volume ofwater that flows per unit time from droplet 1 to droplet 2 across thebilayer can be expressed as (Dixit, S. S., et al., Langmuir 28,7442-7451 (2012) and Cass, A. & Finkelstein, A. J. Gen. Physiol. 50,1765{1784 (1967))

$\begin{matrix}{{{- \frac{dV_{1}}{dt}} = {iPA\overset{¯}{V}{\phi\left( {{C_{2}(t)} - {C_{1}(t)}} \right)}}},} & (9)\end{matrix}$

where P is the permeability coefficient (with dimensions of length perunit time), V is the molar volume of water, ϕ is the osmoticcoefficient, and i is the van't Hoff factor of the salt. Eq. (9)suggests a simple way to estimate the permeability P from experimentalmeasurements:

$\begin{matrix}{\left. {P = {\left\lbrack {iA\overset{¯}{V}{\phi\left( {{C_{1}(0)} - {C_{2}(0)}} \right)}} \right\rbrack^{- 1}\frac{dV_{1}}{dt}}} \right)_{t = 0},} & (10)\end{matrix}$

where C₁(0) and C₂(0) are the initial values of C₁ and C₂, respectively.Because the volume of water is conserved, it may be equivalentlywritten:

$\begin{matrix}{\left. {P = {\left\lbrack {iA\overset{¯}{V}{\phi\left( {{C_{2}(0)} - {C_{1}(0)}} \right)}} \right\rbrack^{- 1}\frac{dV_{2}}{dt}}} \right)_{t = 0}.} & (11)\end{matrix}$

Single droplets containing 1 M KCl were made to form bilayers pairwisewith droplets containing 250 mM KCl, and three such pairs werephotographed through a microscope at intervals of 2 min over a periodof >2 h (FIG. 16A). The droplet and bilayer diameters were measuredusing the ImageJ software package, and the volumes of the dropletscalculated from these diameters by assuming that each droplet had thegeometry of a spherical cap. The initial rates of change of V₁ and V₂for each pair were calculated independently from the first two volumemeasurements. The permeability was then calculated according to Eq. (10)or Eq. (11), assuming V=18.0 ml mol-1, i=2 for KCl, and ϕ=0.90 from theliterature (Hamer, W. J. et al., J. Phys. Chem. Ref. Data 1, 1047-1100(1972)).

9 Model of Folding Networks 9.1 Formulation of the Model

The qualitative behaviour of spontaneously folding networks can bereproduced by a simple model that consists of two coupled components: amechanical part, which models the motion of droplets in a network, andan osmotic part, which models the transfer of water between droplets.

Mechanical Component

Droplets are treated as point masses with an associated radius. If apair of droplets of radii R_(i) and R_(j) approach each other within adistance R_(i)+R_(j), they become connected by a Hookean spring ofnatural length L(R_(i)+R_(j)). The spring represents the bilayer formedbetween the two droplets, and the parameter L<1 approximates thedeformation of the droplets caused by their adhesion.

Given that the characteristic timescale of folding in the experimentswas on the order of minutes to hours, it was estimated that the Reynoldsnumber during folding was on the order of 10⁻⁵. Droplets may thereforebe expected to obey Stokes' law, and a drag force is imposed on eachdroplet that is proportional to its velocity. The net force on eachdroplet i is therefore given by

$\begin{matrix}{{{\overset{\rightarrow}{F}}_{i} = {{\sum\limits_{j \in C_{i}}{{k\left( {r_{ij} - L} \right)}{\hat{\overset{\rightarrow}{r}}}_{ij}}} - {\gamma{\overset{\rightarrow}{v}}_{i}}}},} & (12)\end{matrix}$

where C_(i) is defined as the set of droplets connected to the ithdroplet, {right arrow over (r)}_(ij) is the vector from droplet i todroplet j with magnitude r_(ij), k is the spring constant, γ is adamping coefficient, and v, is the velocity of the ith droplet. Theposition of each droplet i through time, {right arrow over (r)}_(i)(t),is then calculated using

$\begin{matrix}{{{\overset{\rightarrow}{F}}_{i} = {m\frac{d^{2}{\overset{\rightarrow}{r}}_{i}}{dt^{2}}}},} & (13)\end{matrix}$

where m is the mass of the droplet. For reasons discussed below, theinventors assume that the mass, spring constant and damping coefficientsare constant and identical for all droplets.

Osmotic Component

The osmotic component simulates the exchange of water between dropletsof different osmolarities according to Fick's first law. The volume ofwater transferred per unit time from a droplet i with osmolarity C_(i)to a droplet j with osmolarity C_(j) was calculated as

J _(ij) =A _(ij) D(C _(j) −C _(i)),  (14)

where A_(ij) is the area of the bilayer between the two droplets, andthe parameter D represents a permeability coefficient that was assumedto be constant and identical for all bilayers.

9.2 Parameter Values

Table 2 lists the dimensionless values of the parameters used in themodel. The values of D, m and k were chosen to make the timescale ofwater transfer slow compared to the timescale of mechanical relaxation,because the experimentally observed timescales for these processes were,respectively, tens of minutes and a few seconds. The value of γ waschosen to prevent oscillations of the springs while maintaining arelatively short mechanical relaxation time, because droplets are notexperimentally observed to oscillate upon adhesion. C_(low) and C_(high)represent the lower and higher osmolarities of the droplets in thenetwork. The ratio C_(high)/C_(low) was chosen to approximate theexperimental ratio of osmolarities. The remaining variables were chosento reduce the computation time while maintaining numerical stability.Because the timescales of mechanical relaxation and water transfer werechosen so that folding takes place in a mechanical quasi-equilibrium,the simplifying assumption of identical and constant values of m and γfor all droplets, and of k for all bilayers, is not expected to have asignificant effect on the simulations.

TABLE 2 Parameters used in folding model. The parameters D, m and k werechosen to make water transfer take place on a much longer timescale thanmechanical equilibration of the network. The value of y was chosen toprevent oscillations of the springs. C_(high)/C_(low) was chosen toreflect the experimental ratio of osmolarities between the two types ofdroplets. C_(high) − C_(low), Δt and V₀ were chosen to reduce thecomputation time while maintaining numerical stability. Parameter SymbolValue Rate of water transfer D 2 × 10⁻³ Lower osmolarity C_(low)  1Higher osmolarity C_(high) 10 Initial droplet volume V₀ 10 Droplet massm   0.2 Spring constant k  10³ Time step Δt    10⁻² Damping γ   1.1

9.3 Solving the Equations

A computer program was written to simulate the behaviour of the systemas follows. The program reads a series of images that define the networkto be simulated, similarly to the printing program. The droplets areinitially positioned according to the images in a hexagonal close-packedarrangement, which approximates the observed packing of droplets inprinted networks. The droplets are then allowed to equilibratemechanically without exchanging water, to ensure that any motionsubsequently simulated is not due to mechanical equilibration of thedroplets from their initial arrangement. Once this equilibration iscomplete, the osmotic component of the model is activated. At each timepoint t, the program performs the following calculations:

1. Any two droplets i and j that have come within a distance R_(i)+R_(j)are connected by a spring of natural length L(R_(i)+R_(j)).

2. If the osmotic component is active, the volume of water transferredbetween each pair of droplets joined by a bilayer is calculatedaccording to Eq. (14), and the size of each droplet is updatedaccordingly.

3. The position of each droplet at time t+Δt is calculated according toEqs. (12) and (13) using a fourth-order Runge-Kutta scheme.

4. The position of each droplet is updated according to the results ofthe calculations in step 3.

9.4 Visualization

At each time point during the simulation, the program wrote the positionof every droplet to a text file, formatted such that it could be read bythe Visual Molecular Dynamics software package (Humphrey, W., et al., J.Molec. Graphics 14, 33-38 (1996)) to visualize the time evolution of thenetwork. In addition to showing the position and size of each droplet,the visualization colour-coded each droplet according to its osmolarity.The colour scale interpolates from blue to white to red, where blue andred respectively correspond to the lowest and highest osmolarities atthe beginning of the simulation, and white corresponds to the average ofthe two.

Supplementary Discussion 1 Droplet Production

A number of factors other than the amplitude and duration of the voltagepulse were found to significantly affect droplet production by thedroplet generators.

Fluid Levels

Droplet ejection depended strongly on the height difference h betweenthe level of the aqueous phase in the chamber of the droplet generatorand the level of oil solution in the well (FIG. 1), which produced a netforce on the aqueous-oil interface in the nozzle. In general, a greaterheight h yielded larger droplets. Evaporation of the aqueous solutionfrom the chamber gradually changed the aqueous level, so that the sizeof ejected droplets remained consistent for only ˜6 h. Evaporation mightbe prevented by adding a thin layer of oil on top of the aqueous phasein the chamber.

Aqueous Volume

Droplet ejection also depended on the volume of aqueous solution V inthe droplet generator chamber, independently of its height above the oillevel (FIG. 1). In general, the size of ejected droplets was morereadily controlled when the aqueous chamber was completely full thanwhen it was filled incompletely. This is likely because upon theapplication of a voltage pulse to the piezoelectric transducer, aless-filled chamber couples less of the vibration from the transducer tothe fluid in the nozzle, which limits the size of the ejected droplets.

Lipid Concentration

The amplitude of the voltage pulse required to eject droplets of a givensize was lower for higher concentrations of lipid in the oil. This is tobe expected: the adsorption of lipids at the aqueous-oil interface inthe nozzle decreases the tension of that interface, thereby lowering theenergy required to deform the interface to the extent required fordroplet formation.

Nozzle Geometry

Droplets could not be produced reliably in the available range ofvoltages if the nozzle length L was too great, its internal diameter Dwas too small, or its tip diameter d was too great or too small (FIG.1). The dimensions given in FIG. 3 allowed reliable droplet productionwithin the available voltage range. Because the nozzles were producedmanually from glass capillaries, under otherwise almost identicalconditions the two generators generally produced droplets of differentsizes. However, the two generators could be made to eject identicaldroplets by using different voltage pulses for each.

2 Printing Networks

The dynamic viscosity of the oil mixture used was ˜10 times greater thanthat of water. This posed several challenges for the printing of dropletnetworks that do not arise in most other two- and three-dimensionalprinting technologies, which operate in air or aqueous solution:

1. Following the application of a voltage pulse of the piezoelectrictransducer, the pulse of pressure produced at the nozzle creates ashort-lived protrusion of the aqueous phase into the oil phase ˜50-150μm long, which breaks up within tens of ms to create a single droplet.When the nozzle was placed ≲<150 μm from an obstacle, the obstacledeformed the aqueous protrusion, causing the ejected droplet to bedisplaced. Placing the nozzle ≲100 μm from an obstacle precluded dropletformation entirely. Each droplet was therefore ejected ≳200 μm above itsfinal position. However, droplets then required ˜1-5 s to sink fromtheir point of ejection into their intended position in the network.

2. Droplets did not adhere and form a bilayer immediately upon cominginto contact with the network. This is likely to be due to the finitetime required for the oil layer between two droplets to thin under theweight of the falling droplet. Droplets typically formed bilayers withthe network ˜1-3 s after first coming into contact with the network.

3. Droplets not yet incorporated into the network were displaced byviscous shear produced by the motion of the nozzles through the oil.

4. Droplets not yet incorporated into the network were displaced bysubsequent nearby ejections.

In order to print droplets at accurate locations at a rate of ˜1 s⁻¹,the inventors addressed these problems in a series of optimizations.

Nozzle Geometry

The displacement of droplets caused by motion of the nozzle wasminimized by using nozzles with a relatively small outer diameter of˜100 μm. The nozzles were formed from tapered glass capillaries asdescribed in the Supplementary Methods. When using capillaries that hadbeen shaped to have a similar inner diameter but an outer diameter of1.5 mm, the motion of the nozzle displaced the droplets by asignificantly greater distance.

Row Delay

The use of a delay after printing each row of droplets, instead of afterejecting each droplet, allowed a significant reduction in printing timewithout a significant cost to print quality. Although the droplets ineach row were then displaced by motion of the nozzle and subsequentejections in that row, every droplet suffered approximately the samedisplacement, so that droplets were placed in the correct relativepositions within their row. Depending on the orientation of the nozzles,the alternating direction of the printing path for alternating rowscould result in a misalignment of alternate rows. However, themisalignment was easily corrected with a programmed offset for alternaterows (Supplementary Methods).

Sinking Rate

To reduce the row delay necessary to prevent droplet displacement, theinventors increased the rate at which droplets sank through the oil intwo ways. First, the inventors used a salt concentration of 1 M KCl inthe aqueous phase, which increased the density of the droplets relativeto the oil solution. Second, they used droplets ≳30 μm in diameter,because larger droplets suffer proportionately less viscous drag fortheir weight.

Lipid Concentration

For a given concentration of lipid in the oil, smaller droplets requireless time to acquire a lipid monolayer (Kankare, J. et al., Langmuir 15,5591-5599 (1999)). This is because the adsorption of lipid onto thedroplet surface creates a lipid-depleted region around the droplet, andfor smaller droplets this depleted volume is more accessible toreplenishment by transfer from the lipid bath. The droplet diameter istherefore a compromise between a high sinking rate (which occurs withlarger droplets) and a short incubation time (which requires smallerdroplets). In the experiments it was found that droplet diameters in therange ˜30-60 μm were a good compromise.

3 Printing in Bulk Aqueous Solution Printing Process

In contrast to the networks printed in bulk oil, the networks in bulkaqueous solution were printed by controlling the droplet generators andmanipulator during printing using a custom-written graphical userinterface (Supplementary Methods). Because the recipient oil drop usedto print in bulk aqueous solution had a curved lower surface, dropletsejected inside the oil drop rolled down the surface to minimize theirgravitational energy. Therefore the accurate printing of definednetworks in bulk aqueous solution requires knowledge of the geometry ofthe surface of the oil drop. Further, the droplet network design andprinting path should be compatible with that geometry. For instance, foroil drops with the geometries used in these Examples, the aqueousdroplets could be prevented from rolling away from their intendedpositions by printing each layer in an outwardly growing spiral patterncentred about the minimum point.

Geometry of Wire Frames and Networks

The geometry of the oil drop is partially determined by the wire frameused to suspend the drop in bulk aqueous solution. However, the geometryof the drop changes as aqueous droplets are ejected into it, while thevolume of oil remains constant. A network printed in an oil drop maytherefore have a different geometry to the initial geometry of the oildrop, and may have a volume greater than that of the oil drop.

Further, unlike the oil drop, the geometry of a printed network need nothave the minimal bounding surface area, as is evident from the thirdmicrograph in FIG. 11D. This is possible firstly because therearrangement of the network into a configuration with the minimalbounding area would involve the separation of bilayers into monolayers,which is energetically unfavourable (Villar, G., et al., NatureNanotech. 6, 803-808 (2011)) and therefore presents a kinetic barrier.Secondly, the interfacial tension of the bilayer between twoencapsulated droplets is identical or approximately equal to that of thebilayer between an encapsulated droplet and the bulk aqueous solution((Villar, G., et al., Nature Nanotech. 6, 803-808 (2011)), so thethermodynamic stability that would be gained by minimizing the externalarea of the network is likely to be negligible. The geometry of networksprinted in bulk aqueous solution is therefore not fully determined bythe frame. It should also be possible to print networks using frames ina variety of geometries other than simple circular loops, which furtherwidens the possible geometries of networks printed in aqueous solution.

Upper Limits on Oil Volume

The volume of oil that could be stably suspended on a given loopdepended on the diameter of the loop. When a loop was loaded with an oilvolume ≳50% of the volume of a sphere bounded by the loop, the oil dropdeformed over a few minutes until it broke up to form a drop thatfloated upwards in the aqueous phase, while the residual oil remainedattached to the loop. The slow change in geometry of the oil drop waspresumably due to a gradual decrease in surface tension caused by theadsorption of lipid at its surface.

The maximum suspended volume of oil also depended on the geometry of thenozzles. When the two nozzles were inserted into the oil dropapproximately parallel to each other and separated by ≲200 μm, most ofthe oil rose spontaneously in the space between the nozzles. Largerframes may therefore be best designed as fine meshes to prevent the oildrop from breakup instability and from rising between the nozzles bycapillary action.

4 Electrical Recording

It is not possible to determine directly from electrical recordings,such as that in FIG. 12C, whether the measured current stepscorresponded to insertions of αHL into bilayers (i) between droplets inthe network, or (ii) between the network and the large drops impaled byelectrodes. However, the latter is more likely for two reasons, asfollows.

Rate of αHL Insertion

The rate of αHL insertions into droplet interface bilayers decreasedwith time. In droplets that contained αHL in the concentration used inthe electrical recording experiments, the rate of pore insertiondecreased from ˜0.5 s⁻¹ a few minutes after the droplets were formed toa negligible rate after a few hours. The attrition of the rate of poreinsertion is likely to have two contributions. First, droplets were madeby diluting a concentrated stock solution of αHL heptamers solubilizedwith sodium dodecyl sulphate (SDS). The dilution is likely to encouragethe dissociation of SDS from the protein, which would destabilize theheptamers. Further, whereas the stock and diluted solutions of αHL wererefrigerated at −80° C. and 0° C., respectively, the experimental systemwas at room temperature, which would also increase the rate of heptamerdegradation.

The droplets on the conductive pathway were formed from a freshlydiluted solution that was at room temperature during printing for ˜2 hbefore electrical recording, whereas the large drops impaled onelectrodes were formed only ˜15 min before electrical recording. Anypores that inserted in bilayers during electrical recording aretherefore more likely to have been in the large drops than in thedroplets in the pathway.

Consistency of Measured Currents with Electrical Model

The likely configurations of αHL pores in the network was deduced bycomparing the experimentally measured currents with simulations of theelectrical behaviour of droplet networks (FIGS. 14A-14D) (SupplementaryMethods). Only part of the network in FIGS. 12A-12E was simulated todecrease the computation time; the simulation results are not expectedto change significantly with the inclusion of the rest of the network.The measured currents such as in FIG. 12C were consistent with thesimulations only if three conditions were met:

1. Most of the bilayers in the conductive pathway contained at leastseveral αHL pores, so that the pathway had negligible resistancecompared to that of a single pore. This is reasonable, given therelatively high concentration of αHL used and the relatively long timeavailable for pores to insert into bilayers in the pathway.

2. Many of the bilayers between the pathway and one of theelectrode-impaled drops (drop a in FIG. 14A) contained at least one αHLpore, while no pores were present in any of the bilayers that joined thepathway to the other electrode-impaled drop (drop b in FIG. 14A). Thisis plausible, given that one of the large drops was placed onto thenetwork approximately tens of seconds before the other.

3. The bilayers formed by the second electrode-impaled drop to beconnected to the pathway (drop b in FIG. 14A) initially contained no αHLpores, and the first current step corresponded to the insertion of apore in any bilayer between that drop and the pathway. Each subsequentstep was due to the insertion of additional pores in these bilayers.This condition is reasonable, because the experimental electricalrecording began immediately after the second electrode-impaled drop wasjoined to the pathway.

Under these conditions, the simulated current steps closely matched themeasured currents in both the step amplitudes and relaxation timescale(FIG. 14B).

Similarly, the transient current peaks measured with one drop on thepathway and one off the pathway (FIG. 12D) were consistent with thesimulations if, in addition to the above conditions, a thin layer ofdroplets was added that did not allow the flow of current between oneelectrode-impaled drop and the rest of the network. These insulatingdroplets represent the droplets in the network in FIG. 12D that did notcontain αHL. The large drop chosen to be adjacent to this insulatinglayer was drop b in FIG. 14C, which represents the drop that was removedfrom the network and replaced away from the pathway in FIG. 12D. Underthese conditions, both the amplitudes and relaxation time-dependence ofthe current peaks in the simulation (FIG. 14D) were consistent with thetransient currents measured experimentally (FIG. 12E).

5 Self-Folding Networks

This section details the conditions that were found to allow successfolding of droplet networks.

Printing Substrate

Static networks in bulk oil were printed on glass, which was adhesive tothe aqueous droplets, to prevent any unwanted displacement of thenetwork during printing. Conversely, for droplet networks to deformfreely it was found necessary to print on a surface of poly(methylmethacrylate), which was not adhesive to droplets. When printingnetworks on this surface, it was necessary to ensure that the printingsurface was close to horizontal, to prevent the growing network fromdrifting along the surface during printing. The nozzles were alsopositioned ˜150 μm higher from the printing substrate to prevent theejection of droplets from displacing the network.

Timescales of Printing and Folding

If the network folds significantly before printing has completed, thelater droplets may incorporate into the network at incorrect positions.The extent to which the network folds during printing can be minimizedby decreasing the printing time, and increasing the folding timescale.As discussed in an earlier section, the printing time can be shortenedby including a delay after each row rather than after each droplet, andby using larger droplets.

The folding timescale can be increased in two ways:

1. The rate of water transfer between two droplets is proportional tothe difference in their salt concentrations; the ratio of saltconcentrations partially determines the total volume of watertransferred, and therefore the equilibrium geometry of the foldednetwork. The inventors therefore slowed the folding process by reducingthe initial difference in salt concentrations between the two droplettypes, while maintaining a high ratio of initial salt concentrations.

2. The folding timescale was increased further by printing with largerdroplets. Consider a pair of droplets joined by a bilayer, both withinitial volume V but each with a different initial osmolarity. Theinitial rate of water transfer is proportional to the bilayer area, A.Because A∝V^(2/3), water is transported more rapidly between largerdroplets. However, the rate of water transfer as a proportion of thedroplet volume is ∝A/V∝V^(−1/3). Therefore, relative to their initialvolume, larger droplets change in volume more slowly, and by extension anetwork composed of larger droplets will fold more slowly.

Buckling

As the droplets of higher osmolarity swell and those of lower osmolarityshrink in a spontaneously folding network, a length mismatch can developbetween connected regions of the network with different osmolarities.Networks that are thin in one dimension can resolve this mismatchwithout a significant energetic cost by buckling into that dimension. Itwas found that networks designed to fold in the horizontal plane wereless prone to buckle out of that plane if printed with additionalvertical layers. This is presumably because bending the thick networkout of the plane would involve the unfavourable exposure of moremonolayer area or total surface area.

Fracture

In networks that are thick along the axis about which folding takesplace (for example, the axis perpendicular to the page in FIG. 15C), thelength mismatch between connected regions discussed above can producestresses that detach neighbouring shrinking droplets from each other(FIGS. 17A-17C). The deformation of a spontaneously folding networkrequires that the swelling and shrinking portions of the network beconnected both to each other and within themselves, so the fracture ofthe network at some point can preclude further deformation around thatpoint. It was found that this type of fracture did not occur if theshrinking region of the network was made thicker along the axisperpendicular to the interface between the swelling and shrinkingregions (for example, the vertical axis in FIG. 15D, and the horizontalaxis on the page in FIGS. 17A, 17B). This extra thickness presumablydistributes the stresses induced by folding among a greater number ofbilayers, so that the forces on each connected pair of shrinkingdroplets are no longer sufficient to separate the droplets.

CONCLUSIONS

The inventors have shown that the apparatus of the invention can be usedto produce a droplet assembly. The results and methods discussed aboveshow how the apparatus can be adapted to produce the required dropletassembly. Further, the results show that the process of the inventionproduces droplet assemblies with precision and control. The resultsdemonstrate that millimetre-sized networks can be printed. Largernetworks may also be produced. Further, the assemblies canfunctionalised. The inventors have also demonstrated that networks canbe printed to self-fold in predictable ways.

The use of membrane proteins such as aquaporins or ion channels could beused in droplet assemblies to afford greater control over the flow ofwater within droplet networks. Further, the osmotic shape change may bemade reversible, for instance by using osmolytes that are responsive toheating or illumination. Such droplet networks could be developed as ahydraulic mimic of muscle tissue, and may allow the construction ofdroplet networks capable of locomotion.

Because neighbouring cells in a living tissue are separated by twomembranes, they must communicate through specialized membrane proteinssuch as gap junctions (Nakagawa, S., et al., Curr. Opin. Struc. Biol.20, 423-430 (2010)) to cooperate, and so produce the emergent propertiesthat distinguish a tissue from a collection of independently functioningcells. The system of the present invention, in which a single layer(such as a single lipid bilayer) separates adjacent aqueouscompartments, allows these collective properties to emerge in a simplerenvironment. The automated printing process presented here will allowthe construction of networks that use other membrane proteins andosmolytes to reproduce more sophisticated behaviours. Such dropletnetworks might function autonomously within living organisms or beinterfaced with tissues, for example as platforms for drug delivery, orto augment or replace failing tissues or organs.

1. A droplet assembly which is obtainable by a process for producing adroplet assembly using an apparatus for producing the droplet assembly,which droplet assembly comprises: a plurality of droplets, wherein eachof said droplets comprises: (i) a droplet medium, and (ii) an outerlayer of amphipathic molecules around the surface of the droplet medium,wherein the droplet medium is an aqueous medium or a hydrophobic medium,and wherein at least one of said droplets contacts another of saiddroplets to form a layer of said amphipathic molecules as an interfacebetween the contacting droplets; which apparatus comprises: at least onedroplet generator; a container which is moveable relative to the atleast one droplet generator; and a control unit, which control unit isadapted to control the dispensing of droplets from the at least onedroplet generator and the movement of the container relative to the atleast one droplet generator; wherein said container of the apparatuscontains a bulk medium, wherein: when the droplet medium is an aqueousmedium the bulk medium is a hydrophobic medium, and when the dropletmedium is a hydrophobic medium the bulk medium is an aqueous medium;which process comprises: (a) a plurality of dispensing steps, whereineach dispensing step comprises dispensing a droplet of the dropletmedium from a said droplet generator into the bulk medium, in thepresence of amphipathic molecules, and thereby forming in the bulkmedium a droplet which comprises (i) said droplet medium and (ii) anouter layer of amphipathic molecules around the surface of the dropletmedium; and (b) moving the container relative to the at least onedroplet generator, to control the relative positioning of the dropletsin the bulk medium.
 2. A droplet assembly which comprises a plurality ofdroplets, wherein each of said droplets comprises (i) an aqueous medium,and (ii) an outer layer of amphipathic molecules around the surface ofthe aqueous medium, and wherein each of said droplets contacts anotherof said droplets to form a bilayer of said amphipathic molecules as aninterface between the contacting droplets, wherein the plurality ofdroplets comprises a first region of said droplets and a second regionof said droplets, wherein each droplet in the first region contacts atleast one other droplet in the first region to form a bilayer of saidamphipathic molecules as an interface between the contacting droplets,and each droplet in the second region contacts at least one otherdroplet in the second region to form a bilayer of said amphipathicmolecules as an interface between the contacting droplets, wherein theaqueous medium of the droplets in the first region has a firstosmolarity and the aqueous medium of the droplets in the second regionhas a second osmolarity, wherein the first osmolarity is different fromthe second osmolarity.
 3. The droplet assembly according to claim 2,wherein the ratio of the first osmolarity to the second osmolarity isfrom 2:1 to 50:1, preferably from 5:1 to 20:1.
 4. The droplet assemblyaccording to claim 2, wherein the aqueous medium of the droplets in thefirst region is an aqueous solution of a salt, which salt has a firstconcentration in the aqueous solution, and the aqueous medium of thedroplets in the second region is an aqueous solution of the same salt,which salt has a second concentration in the aqueous solution, whereinthe first concentration is different from the second concentration. 5.The droplet assembly according to claim 4, wherein the ratio of thefirst concentration to the second concentration is from 2:1 to 50:1,preferably from 5:1 to 20:1.
 6. The droplet assembly according to claim2, wherein the droplets in the first region are arranged in a row, aplurality of rows, a layer or a plurality of layers, and the droplets inthe second region are arranged in a row, a plurality of rows, a layer ora plurality of layers.
 7. The droplet assembly according to claim 2,wherein the droplets in the first region are arranged in a layer or aplurality of layers, and the droplets in the second region are arrangedin a layer or a plurality of layers, wherein droplets in the secondlayer or plurality of layers are disposed on or adjacent to droplets inthe first layer or plurality of layers, so that droplets in the firstlayer contact droplets in the second layer to form bilayers of saidamphipathic molecules as interfaces between the contacting droplets. 8.The droplet assembly according to claim 7, wherein the first and secondlayers or pluralities of layers comprise petal-shaped regions.
 9. Thedroplet assembly according to claim 8, wherein the petal-shaped regionsare capable of folding inwards and joining to form a hollow dropletassembly.
 10. A droplet assembly which comprises a plurality ofdroplets, wherein each of said droplets comprises (i) an aqueous medium,and (ii) an outer layer of amphipathic molecules around the surface ofthe aqueous medium, and wherein each of said droplets contacts anotherof said droplets to form a bilayer of said amphipathic molecules as aninterface between the contacting droplets, wherein the plurality ofdroplets defines a shell around a volume within the droplet assemblythat does not comprise said droplets.
 11. The droplet assembly accordingto claim 10, wherein the shell defined by said droplets is a curvedstructure.
 12. The droplet assembly according to claim 10, wherein theshell defined by said droplets is substantially cylindrical,substantially ring-shaped, substantially spherical, or substantiallyhemispherical.
 13. The droplet assembly according to claim 11, whereinthe shell defined by said droplets encloses said volume within thedroplet assembly.
 14. The droplet assembly according to claim 13,wherein the shell defined by said droplets is substantially spherical.15. The droplet assembly according to claim 1, wherein the number ofsaid droplets in the plurality of droplets is at least 10,000.